Chemically-enhanced primer compositions, methods and kits

ABSTRACT

A chemically-enhanced primer is provided comprising a negatively charged moiety (NCM), an oligonucleotide sequence having a) non-nuclease resistant inter-nucleotide linkages or b) at least one nuclease resistance inter-nucleotide linkage. The chemically-enhanced primer can be used for sequencing and fragment analysis. Methods for synthesizing the chemically-enhanced primer as well as a method of preparing DNA for sequencing, a method of sequencing DNA, and kits containing the chemically-enhanced primer are also provided. The method of sequencing DNA can comprise contacting amplification reaction products with the composition wherein excess amplification primer is degraded by the nuclease and the chemically-enhanced primer is essentially non-degraded.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims priority fromU.S. application Ser. No. 13/284,839, filed Oct. 28, 2011, which claimspriority from U.S. Provisional Patent Application No. 61/407,899, filedOct. 28, 2010 and U.S. Provisional Patent Application No. 61/408,553,filed Oct. 29, 2010, each of which are incorporated herein by reference.

FIELD

The present teachings pertain to chemically modified oligonucleotidesequence primer compositions and methods for sequencing DNA and fragmentanalysis. The teachings also relate to compositions for preparing,fragment analysis and sequencing of nucleic acids such as cDNA and DNA.

BACKGROUND

A standard polymerase chain reaction (PCR)/sequencing workflow generallyincludes five steps requiring reagent addition: an initial PCR step, aPCR clean-up step, a sequencing step, a sequencing cleanup step, andelectrophoresis. The PCR step involves amplification of a templatepolynucleotide using amplification primers and a thermo-stable DNApolymerase enzyme. The PCR cleanup step is commonly done by the additionof exonuclease I and alkaline phosphatase, followed by incubation, andsubsequent heat-inactivated to inactivate the enzymes. A standardPCR/sequencing workflow is illustrated in FIG. 1A.

A typical PCR reaction uses an excess of amplification primers, someprimers remain unincorporated upon completion of the PCR reaction. Thisnecessitates removal of the excess primers before proceeding to asequencing reaction, because the excess amplification primers willinterfere with the subsequent sequencing reaction. The PCR reactionfurthermore contains an excess of dNTPs that can interfere with thesubsequent sequencing reaction. The hydrolytic properties of exonucleaseI which degrades single-stranded DNA present in the PCR mixture allowsthe amplification product (amplicon) to be used more efficiently insubsequent sequencing applications. The enzyme activity of alkalinephosphatase dephosphorylates free dNTPs remaining from the PCR reaction.After an appropriate incubation period, the exonuclease I and alkalinephosphatase enzymes are heat inactivated before adding sequencingprimer, dNTPs, and dye-labeled ddNTPs; otherwise the enzymes woulddegrade these reagents and the sequencing reaction products.

Without adequate exonuclease I treatment to remove excess PCRamplification primers, aberrant sequence ladders can be generated. Anexcess of dNTPs can produce a weak sequencing signal and/or shortsequence reads. The need to obtain high quality sequence results at base1 from the sequencing primer is also often difficult. The transitionfrom amplification to efficient sequencing has made high quality 5′sequence resolution and clean-up of unincorporated dNTPs andamplification primers a priority to obtain clean sequencing results.

Resolution of nucleic acid sequence near the sequencing primer has beendifficult to obtain without sacrificing throughput residence time duringelectrophoresis with POP7™ polymer. Adjustments in the type of mobilitysystem, for example, using the POP6™ polymer matrix, adjustingdenaturing conditions and temperature can improve resolution but alwaysat the expense of increased electrophoresis time as POPE polymerrequires longer electrophoresis time. Difficulties in removal ofunincorporated reactants and long residence time when performingsize-dependent mobility separation contribute to inefficiencies innucleic acid sequencing. A need exists for improved methods for thePCR/sequencing and PCR/fragment analysis workflow and sequenceresolution following PCR amplification.

SUMMARY

In one aspect, the invention provides a chemically-enhanced primercomprising: an oligonucleotide sequence having a) non-nuclease resistantinter-nucleotide linkages or b) at least one nuclease resistanceinter-nucleotide linkage; and a negatively charged moiety (NCM). In someembodiments, the chemically-enhanced primer includes a NCM having astructure of the formula:

where each instance of n is independently an integer of 1 to 9; and x isan integer of 1 to about 30. In some embodiments, thechemically-enhanced primer has a structure of Formula IV:

where each instance of n is independently an integer of 1 to 9; x is aninteger of 1 to 50; v is an integer of 1 to 9; t is 0 or 1; LINKERincludes 3-100 atoms; and OLIGO has a structure of the followingformula:

where B is a nucleobase; K is S or O; m is 0 or 1; z is an integer of 3to about 100; W is OH, F, OMe, or H; and Nt is a moiety having aformula:

In yet other embodiments, a chemically-enhanced primer has a structureof the formula: (Cn)_(x)-OLIGO, wherein (Cn)_(x) has a structure of thefollowing formula:

where each instance of n is independently an integer of 1 to 9; and x isan integer of 1 to about 30; and OLIGO has a structure as defined forFormula IV.

In further embodiments of the chemically-enhanced primer of Formula IV,a chemically-enhanced primer is provided having a structure of thefollowing formula: (Cn)_(x)-OLIGO*, wherein (Cn)_(x) has a structure ofthe following formula:

where each instance of n is independently an integer of 1 to 9; and x isan integer of 1 to about 30; and OLIGO* has a structure of the followingformula:

where B is a nucleobase; W is OH, F, OMe, or H; x is an integer of 1 toabout 30; and z is an integer of 3 to about 100.

In some embodiments of the chemically-enhanced primer of Formula IV, thechemically-enhanced primer is fluorescently labeled. In otherembodiments, the chemically-enhanced primer is not fluorescentlylabeled. The chemically-enhanced primer may have an OLIGO portion whereW is H or OH. The chemically-enhanced primer may have K=S. In otherembodiments, the chemically-enhanced primer may have K=O. In someembodiments of the chemically-enhanced primer, n is 3 or 6. In otherembodiments, each instance of n is independently 3 or 6. In yet otherembodiments, when x is greater than 5, then a first plurality of n isselected to be 3, and a second plurality of n is selected to be 6. Forsome embodiments of the chemically-enhanced primer, x is 5, 8, 9, 10, or15. The chemically-enhanced primer may have z, where z is an integer of5 to 30.

For some embodiments of the chemically-enhanced primer of Formula IV,OLIGO may be a universal primer. In some embodiments, the universalprimer is selected from M13, US1, T7, SP6, and T3. In other embodimentsof the chemically-enhanced primer of Formula IV, OLIGO may be a genespecific oligonucleotide sequence.

For some embodiments of the chemically-enhanced primer of Formula IV,the chemically-enhanced primer is resistant to digestion by a nuclease.In some embodiments, the nuclease is selected from exonuclease I, ExoIII, Pfu and DNA pol I.

In another aspect, the invention provides a chemically-enhanced primerhaving a structure of Formula I:

where B is a nucleobase; K is S or O; each instance of n isindependently an integer of 1 to 9; m is 0 or 1; x is an integer of 1 toabout 30; z is an integer of 3 to about 100; and Nt is a moiety having aformula:

and W is OH, F, OMe, or H.

In some embodiments of the chemically-enhanced primer of Formula I, m is0. In other embodiments, K is S. For yet other embodiments, K is O. Thechemically-enhanced primer may be fluorescently labeled. In otherembodiments, the chemically-enhanced primer is not fluorescentlylabeled. The chemically-enhanced primer may have a structure where W isH or OH.

In some embodiments of the chemically-enhanced primer of Formula I, n is3 or 6. In other embodiments, when x is greater than 5, a firstplurality of n is selected to be 3, and a second plurality of n isselected to be 6. In yet other embodiments, x is 5, 8, 9, 10, or 15. Insome embodiments, z is an integer of 5 to 30.

In some embodiments of the chemically-enhanced primer of Formula I,having OLIGO representing the oligonucleotide portion of the primer,OLIGO may be a universal primer. In some embodiments, the universalprimer is selected from M13, US1, T7, SP6, and T3. In other embodiments,having OLIGO representing the oligonucleotide portion of the primer,OLIGO may be a gene specific oligonucleotide sequence

In some embodiments of the chemically-enhanced primer of Formula I, hasa structure of one of the following formulae:

wherein FL is a dye label and B^(f) is a dye labeled nucleobase.

In another aspect, a composition is provided for sequencing nucleic acidcomprising: a chemically-enhanced primer of Formula I:

wherein B is a nucleobase; K is S or O; each n is independently aninteger of 1 to 9; m is 0 or 1; x is an integer of 1 to about 30; z isan integer of 3 to about 100; W is OH, F, OMe, or H; and Nt is a moietyhaving a formula:

In some of the embodiments of the composition for sequencing nucleicacid, the chemically-enhanced primer of Formula I has a structure havinga formula of one of Formula I-A, Formula I-B, Formula I-C, Formula I-D,Formula I-E, Formula I-F, Formula I-G, Formula I-H, Formula I-J, orFormula I-K.

In some of the embodiments of the composition for sequencing nucleicacid, the oligonucleotide sequence portion of the chemically-enhancedprimer may be a universal primer. In some embodiments, the universalprimer is selected from M13, US1, T7, SP6, and T3. In furtherembodiments, the universal primer is M13. In some embodiments, thechemically-enhanced primer may include one nuclease-resistant linkage.

In some of the embodiments of the composition for sequencing nucleicacid, the composition further includes a polymerase, a nuclease,deoxynucleotide triphosphates, dideoxynucleotide triphosphates and adye-label. In other embodiments, the composition further includes apolymerase, deoxynucleotide triphosphates, dideoxynucleotidetriphosphates and a dye-label. In some embodiments, thedideoxynucleotide triphosphates may comprise dye-labeleddideoxynucleotide triphosphates. The dye-labeled dideoxynucleotidetriphosphates may comprise fluorescent dye-labeled dideoxynucleotidetriphosphates. In other embodiments, the dye-label is attached to theNCM or the oligonucleotide sequence.

In some of the embodiments of the composition for sequencing nucleicacid, when a nuclease is present, the nuclease is selected fromexonuclease I, Exo III, Pfu and DNA pol I. The composition forsequencing nucleic acid may further comprise a PCR amplificationreaction product that includes non-nuclease-resistant amplificationprimer(s). The PCR amplification reaction product may further comprisean amplified DNA target sequence.

In another aspect, a method is provided to prepare DNA for sequencing,comprising the steps of: amplifying the DNA under conditions to produceamplification reaction products, the amplification reaction productscomprising excess amplification primer; and contacting the amplificationreaction products with a reaction mixture comprising a nuclease and achemically-enhanced primer, whereby the excess amplification primer isdegraded by the nuclease and the chemically-enhanced primer isessentially non-degraded, wherein the chemically-enhanced primerincludes at least one negatively charged moiety (NCM) and anoligonucleotide sequence having a) non-nuclease resistantinter-nucleotide linkages or b) at least one nuclease resistanceinter-nucleotide linkage.

In some of the embodiments of the method for preparing DNA forsequencing, the amplification reaction products further comprise atarget amplicon. In some of the embodiments of the method for preparingDNA for sequencing, the chemically-enhanced primer includes onenuclease-resistant linkage at a terminal 3′ end of the primer. In otherembodiments, the NCM includes one or more negatively charged moietieseither at a terminal 5′ end.

In another aspect, a method is provided to sequence DNA, comprising thesteps of: reacting DNA in a sequencing reaction wherein thechemically-enhanced primer primes the sequencing reaction.

In another aspect, a method is provided to sequence DNA, comprising thesteps of: amplifying DNA in a first reaction mixture comprisingnuclease-sensitive amplification primers to form amplified DNA;contacting the first reaction mixture of the amplifying step with asecond reaction mixture comprising a nuclease and a chemically-enhancedprimer whereby the nuclease sensitive amplification primers are degradedby the nuclease; inactivating the nuclease; and reacting the amplifiedDNA in a sequencing reaction wherein the chemically-enhanced primerprimes the sequencing reaction.

In some of the embodiments of the methods for sequencing DNA, the methodfurther includes the steps of: obtaining sequencing results based on thesequencing reaction; and determining a nucleotide base sequence of theamplified DNA based on the results. In other embodiments, the step ofamplifying DNA includes the step of polymerase chain reactionamplification. In yet other embodiments, the sequencing reactionincludes cycle sequencing. In some embodiments, the nuclease is selectedfrom exonuclease I, Exo III, Pfu and DNA pol I.

In some of the embodiments of the methods for sequencing DNA, the secondreaction mixture further includes a polymerase, deoxynucleotidetriphosphates, dideoxynucleotide triphosphates and a dye-label.

In some of the embodiments of the methods for sequencing DNA, thechemically-enhanced primer includes an oligonucleotide sequence havinga) non-nuclease resistant inter-nucleotide linkages or b) at least onenuclease resistance inter-nucleotide linkage and a NCM. In someembodiments, the NCM includes a moiety having a structure of thefollowing formula:

wherein each n is independently an integer of 1 to 9, and x is aninteger of 1 to about 30. In some embodiments, the chemically-enhancedprimer has a structure of one of Formula I, Formula I-A, Formula I-B,Formula I-C, Formula I-D, Formula I-E, Formula I-F, Formula I-G, FormulaI-H, Formula I-J, Formula I-K, Formula II, Formula III, Formula IV,(Cn)_(x)-OLIGO, (Cn)_(x)-OLIGO*, Formula V, Formula V-A, Formula VI,Formula VI-A, or Formula VI-A1, as disclosed here.

In some of the embodiments of the methods for sequencing DNA, thechemically-enhanced primer includes one nuclease-resistant linkage at aterminal 3′ end. In other embodiments, the chemically-enhanced primerincludes a plurality of NCMs either at a terminal 5′ end or within aoligonucleotide sequence of the chemically-enhanced primer. In someother embodiments, the NCM includes a plurality of (Cn) spacers. Inother embodiments, n equals 3, the NCM includes a (C3)_(x) spacer,wherein x equals at least 5, at least 6, at least 8, at least 9, atleast 10, at least 15 at least 18, at least 20, at least 24 or more C3spacers in a linear or branched arrangement. In other embodiments, theplurality of (Cn) spacers may be doubler or a trebler.

In yet another aspect, a method is provided to resolve sequencingambiguity comprising the steps of: amplifying DNA suspected ofcomprising an ambiguity in a first reaction mixture comprisingnuclease-sensitive amplification primers to form amplified DNA;contacting the first reaction mixture of the amplifying step with asecond reaction mixture comprising a nuclease and a chemically-enhancedprimer, whereby the nuclease sensitive amplification primers aredegraded by the nuclease; inactivating the nuclease; and reacting theamplified DNA in a sequencing reaction wherein the chemically-enhancedprimer primes the sequencing reaction. In some embodiments, wherein theDNA is an HLA gene, an HLA allele, an oncogene or a sequence comprisinga polymorphism.

In a further aspect, a system is provided to sequence DNA comprising:amplifying DNA in a first reaction mixture comprising nuclease sensitiveamplification primers to form amplified DNA; contacting the firstreaction mixture of the amplifying step with a second reaction mixturecomprising a nuclease and a chemically-enhanced primer, whereby thenuclease sensitive amplification primers are degraded by the nuclease;inactivating the nuclease; reacting the amplified DNA in a sequencingreaction wherein the chemically-enhanced primer primes said sequencingreaction; and identifying a nucleotide base sequence of the amplifiedDNA by mobility-dependent separation of sequencing reaction products.

In some embodiments of the system for sequencing DNA, themobility-dependent separation is selected from separation by charge andseparation by size. In some embodiments, the separation by size pluscharge is selected from gel electrophoresis and capillaryelectrophoresis. In other embodiments, the separation by size isselected from a liquid gradient and a denaturing gradient.

In yet a further aspect, a kit is provided including achemically-enhanced primer. In some embodiments, the chemically-enhancedprimer includes a NCM and an oligonucleotide sequence having a)non-nuclease resistant inter-nucleotide linkages or b) at least onenuclease resistance inter-nucleotide linkage. In other embodiments, theNCM includes a moiety having a structure of the following formula:

where each n is independently an integer of 1 to 9, and x is an integerof 1 to about 30. In some embodiments, the chemically-enhanced primer ofthe kit has a structure of one of Formula I, Formula I-A, Formula I-B,Formula I-C, Formula I-D, Formula I-E, Formula I-F, Formula I-G, FormulaI-H, Formula I-J, Formula I-K, Formula II, Formula III, Formula IV,(Cn)_(x)-OLIGO, (Cn)_(x)-OLIGO*, Formula V, Formula V-A, Formula VI,Formula VI-A, or Formula VI-A1, as disclosed here. In some embodiments,the chemically enhanced primer has an oligonucleotide sequence selectedfrom the group consisting of Cn)_(x)-US1, (Cn)_(x)-M13-forward,(Cn)_(x)-M13-reverse, (Cn)_(x)-T7, (Cn)_(x)-SP6, and (Cn)_(x)-T3. Insome embodiments, the chemically enhanced primer the chemically-enhancedprimer is (Cn)_(x)-GSO, wherein GSO is a gene specific oligonucleotidesequence, and includes 50 or fewer nucleotides.

In some embodiments of the kit, the kit further includes at least one ofinstructions for use, a nuclease, a sufficient quantity of enzyme for asequencing reaction or a fragment analysis reaction, buffer tofacilitate the sequencing reaction or fragment analysis reaction, dNTPs,modified dNTPs, dNTP analogs and 7-Deaza-dGTP for strand extensionduring sequencing reaction or fragment analysis reaction, ddNTPs, adye-label, loading solution for preparation of the sequenced material orfragment analysis material for electrophoresis, genomic DNA as atemplate control, a size marker to insure that materials migrate asanticipated in the separation medium, and a protocol and manual toeducate the user and limit error in use. In other embodiments, the kitfurther includes at least one of instructions for use, a sufficientquantity of enzyme for a sequencing reaction or a fragment analysisreaction, buffer to facilitate the sequencing reaction or fragmentanalysis reaction, dNTPs, modified dNTPs, dNTP analogs and 7-Deaza-dGTPfor strand extension during sequencing reaction or fragment analysisreaction, ddNTPs, a dye-label, loading solution for preparation of thesequenced material or fragment analysis material for electrophoresis,genomic DNA as a template control, a size marker to insure thatmaterials migrate as anticipated in the separation medium, and aprotocol and manual to educate the user and limit error in use. In otherembodiments,

According to various embodiments, the present teachings provide acomposition for a chemically-enhanced primer. According to variousembodiments, the primer can comprise a negatively charged moiety, anoligonucleotide sequence and a nuclease-resistant linkage. In variousembodiments the primer can be used in fragment analysis, sequencingnucleic acid and for improving resolution, PCR through sequencingworkflows with POP-7™ polymer on capillary electrophoresis instrumentssuch as those manufactured by Applied Biosystems (Foster City, Calif.).

According to various embodiments, the present teachings provide acomposition for sequencing nucleic acid. According to variousembodiments, the composition can comprise a composition comprising achemically-enhanced primer comprising an oligonucleotide sequence, anegatively charged moiety (NCM) and at least one nuclease-resistantlinkage. In various embodiments the composition can further comprise apolymerase, a nuclease, deoxynucleotide triphosphates (dNTPs), anddideoxynucleotide triphosphates (ddNTPs) and at least one dye-label. Invarious embodiments of the method, the composition can be added in onestep directly to a PCR reaction product, without first removing excessPCR amplification primers from the PCR reaction product.

According to various embodiments, the present teachings relate to amethod of preparing DNA for sequencing, a method of sequencing DNA, anda composition for sequencing DNA. The teachings provide a method ofPCR/sequencing (including cycle sequencing) that can be quicker andsimpler, and require fewer steps, than traditional methods. The methodsof the present teachings utilize a chemically-enhanced primer incombination with nuclease, which can reduce sequence noise and removeundesired sequence priming. The present teachings further provide acomposition for DNA sequencing that can be used with such a method.

According to various embodiments, the present teachings disclose amethod of preparing DNA for sequencing. In some embodiments, the DNApreparation method can eliminate at least one reagent addition step usedin conventional PCR/cycle sequencing, thereby reducing the number ofprocessing steps.

According to various embodiments, a method of preparing DNA forsequencing is provided that can comprise amplifying DNA under conditionsto produce amplification reaction products, the amplification reactionproducts comprise excess amplification primer, and contacting theamplification reaction products with a reaction mixture comprising anuclease and a chemically-enhanced sequencing primer, under conditionsin which the excess amplification primer is degraded by the nuclease.According to various embodiments, the chemically-enhanced primer isessentially non-degraded under such conditions. In some embodiments, theexcess amplification primer can comprise inter-nucleotide phosphodiesterbonds that are susceptible to nuclease cleavage. In some embodiments thechemically-enhanced primer can comprise at least one inter-nucleotidenuclease-resistant linkage, including but not limited to at least onephosphorothioate bond that is not susceptible to nuclease cleavage.

The present teachings further provide a method of sequencing DNA thatcan generate clean, clear and accurate sequencing data by a simplerworkflow compared to conventional methods, and that requires less time.According to various embodiments, a DNA sequencing method is providedthat can comprise adding a sequencing reaction mix directly to acompleted PCR amplification reaction, without first performing aseparate cleanup step; that is, without first removing excess PCRamplification primers by the addition of a nuclease and completing anuclease inactivation step, followed by a second addition of sequencingprimers and reagents.

According to various embodiments, a method of sequencing DNA is providedthat can comprise amplifying DNA in a first reaction mixture comprisingnuclease-sensitive amplification primers to form amplified DNA,contacting the first reaction mixture with a second reaction mixturecomprising a nuclease and a chemically-enhanced primer under conditionsin which the nuclease-sensitive amplification primers are degraded bythe nuclease, inactivating the nuclease, and causing the amplified DNAto serve as template in a sequencing reaction under conditions in whichthe chemically-enhanced primer primes the sequencing reaction.

The present teachings further provide a system for sequencing DNA thatcan comprise amplifying DNA in a first reaction mixture comprisingnuclease sensitive amplification primers to form amplified DNA,contacting said first reaction mixture of the amplifying step with asecond reaction mixture comprising a nuclease and a chemically-enhancedprimer, under conditions in which the nuclease sensitive amplificationprimers are degraded by the nuclease; inactivating the nuclease andcausing the amplified DNA to react in a sequencing reaction underconditions in which the chemically-enhanced primer primes saidsequencing reaction; and identifying a nucleotide base sequence of theamplified DNA by mobility-dependent separation of sequencing reactionproducts.

The present teachings further provide a kit. In various embodiments thekit comprises a chemically-enhanced primer comprising a negativelycharged group, an oligonucleotide sequence and a nuclease resistantmoiety. In further embodiments, the kit can have at least one of ainstructions for use, a nuclease, a sufficient quantity of enzyme forsequencing or fragment analysis, buffer to facilitate the sequencing orfragment analysis, dNTPs, modified dNTPs, dNTP analogs and 7-Deaza-dGTPfor strand extension during sequencing or fragment analysis, ddNTPs, adye-label, loading solution for preparation of the sequenced or fragmentanalyzed material for electrophoresis, genomic DNA as a templatecontrol, a size marker to insure that materials migrate as anticipatedin the separation medium, and a protocol and manual to educate the userand limit error in use.

Various patents, patent applications, and other publications arereferred to herein, all of which are incorporated herein in theirentireties by reference. In addition, the following standard referenceworks are incorporated herein by reference: Current Protocols inMolecular Biology, John Wiley & Sons, N.Y., edition as of October 2007;Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual,3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001.In the event of a conflict between the instant specification and anydocument incorporated by reference, the specification shall control, itbeing understood that the determination of whether a conflict orinconsistency exists is within the discretion of the inventors and canbe made at any time.

Additional features and advantages of the present teachings will beevident from the description that follows, and in part will be apparentfrom the description, or can be learned by practice of the presentteachings. It is to be understood that both the foregoing summary andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the present teachingswithout limiting the present teachings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification exemplify embodiments disclosed and, togetherwith the description, serve to explain and illustrate principles ofdisclosed embodiments. Specifically:

FIG. 1 is a diagrammatic representation of a standard PCR/cyclesequencing workflow; with five steps in FIG. 1A and four steps in FIG.1B, the disclosed improved workflow. The numbers indicated in each boxrepresents the number of minutes each step requires. It is shown thatuse of the chemically-enhanced primers permit significant reduction inworkflow time.

FIG. 2 illustrates an exonuclease I-resistant oligonucleotide having anuclease-resistant linkage at the terminal 3′ end, according to variousembodiments.

FIG. 3A illustrates a chemically-enhanced primer consisting of(C3)₁₀-M13*(Forward).

FIG. 3B illustrates a chemically-enhanced primer consisting of (C3)₃-M13(Forward).

FIG. 3C illustrates a chemically-enhanced primer consisting of (C3)₉-M13(Forward).

FIG. 3D illustrates a chemically-enhanced primer consisting of (C3)₅-M13(Forward).

FIG. 3E illustrates a chemically-enhanced primer consisting of(C3)₆-trebler-M13 (Forward).

FIG. 3F illustrates a chemically-enhanced primer consisting of(C3)₃-Long trebler-M13 (Forward).

FIG. 3G illustrates a chemically-enhanced primer consisting of(C₃₋)₈-treb-M13 (Forward).

FIG. 3H illustrates a chemically-enhanced primer consisting of(C3)₁₉-M13* (Forward), * indicates a phosphorothioate linkage.

FIG. 3I illustrates a chemically-enhanced primer consisting of(C3)₁₉-M13* (Forward), indicates a phosphorothioate linkage.

FIG. 3J illustrates a chemically-enhanced primer consisting of(C3)₁₅-M13* (Forward), * indicates a phosphorothioate linkage

FIG. 4A-4B illustrates a chemically-enhanced primer consisting of(C3)₁₅-gene specific primer oligonucleotide sequence* (Forward) or auniversal primer oligonucleotide sequence* (Forward), respectively,indicates a phosphorothioate linkage.

FIG. 4C illustrates a chemically-enhanced primer consisting of(C3)₁₅-oligonucleotide sequence (Forward).

DETAILED DESCRIPTION

To facilitate understanding of the present teachings, the followingdefinitions are provided. It is to be understood that, in general, termsnot otherwise defined are to be given their ordinary meanings ormeanings as generally accepted in the art.

As used herein, the term “PCR/cycle sequencing” refers to a method fordetermining a nucleotide sequence of DNA by PCR amplifying the DNA,followed by sequencing reactions repeated (or cycled) several times.This cycling is similar to PCR because the sequencing reaction isallowed to proceed at 42° C.-55° C., then stopped at 95° C., and startedagain at 42° C.-55° C., and uses a thermostable DNA polymerase.

As used herein, the term “phosphorothioate linkage” refers to aninter-nucleotide linkage comprising a sulfur atom in place of anon-bridging oxygen atom within the phosphate linkages of a sugarphosphate backbone. The term phosphorothioate linkage refers to bothphosphorothioate inter-nucleotide linkages and phosphorodithioateinter-nucleotide linkages. A “phosphorothioate linkage at a terminal 3′end” refers to a phosphorothioate linkage at the 3′ terminus, that is,the last phosphate linkage of the sugar phosphate backbone at the 3′terminus. A phosphorothioate linkage at a terminal 3′ end is illustratedin FIG. 2.

As used herein, the term “phosphodiester linkage” may refer to thelinkage —PO₄-which is used to link nucleotide monomers, such as theinter-nucleotide linkages found in naturally-occurring DNA.Additionally, “phosphodiester linkage” may refer to portions of the NCMsor NCM linkers of the chemically-enhanced primers of the presentdisclosure.

As used herein, the term “nuclease-resistant linkage” refers to anoligonucleotide sequence, such as a primer, that is resistant todigestion in the 3′ to 5′ direction by nuclease. Phosphorothioate andboronophosphate linkages are two examples of nuclease-resistantlinkages. The examples are not to be construed as limited to just theseexamples.

As used herein, the term “primer” refers to an oligonucleotide,typically between about 10 to 100 nucleotides in length, capable ofselectively binding to a specified target nucleic acid or “template” byhybridizing with the template. The primer can provide a point ofinitiation for template-directed synthesis of a polynucleotidecomplementary to the template, which can take place in the presence ofappropriate enzyme(s), cofactors, substrates such as nucleotides andoligonucleotides and the like.

As used herein, the term “chemically-enhanced primer” refers to a primerthat can have a negatively charged moiety at a terminal 5′ end of theprimer or within the primer. The primer can also include anuclease-resistant linkage at the last phosphate linkage of the sugarphosphate backbone at the 3′ terminus.

As used herein, the term “sequencing primer” refers to anoligonucleotide primer that is used to initiate a sequencing reactionperformed on a nucleic acid. The term “sequencing primer” refers to botha forward sequencing primer and to a reverse sequencing primer.

As used herein, the term “extension primer” refers to anoligonucleotide, capable of annealing to a nucleic acid region adjacenta target sequence, and serving as an initiation primer for elongation ofthe oligonucleotide by using the target sequence as the complementarytemplate for nucleotide extension under suitable conditions well knownin the art. Typically, a sequencing reaction employs at least oneextension primer or a pair of extension primers. The pair would includean “upstream” or “forward” primer and a “downstream” or “reverse”primer, which delimit a region of the nucleic acid target sequence to besequenced.

As used herein, the term “amplification primer” refers to anoligonucleotide, capable of annealing to an RNA or DNA region adjacent atarget sequence, and serving as an initiation primer for nucleic acidsynthesis under suitable conditions well known in the art. Typically, aPCR reaction employs a pair of amplification primers including an“upstream” or “forward” primer and a “downstream” or “reverse” primer,which delimit a region of the RNA or DNA to be amplified.

As used herein, the term “tailed primer” or “tailed amplificationprimer” refers to a primer that includes at its 3′ end a sequencecapable of annealing to an RNA or DNA region adjacent a target sequence,and serving as an initiation primer for DNA synthesis under suitableconditions well known in the art. The primer includes its 5′ end asequence capable of annealing to a sequencing primer, for example, anoligonucleotide sequence, an universal sequencing primer, agene-specific primer, primer and the like.

As used herein, the term “amplifying” refers to a process whereby aportion of a nucleic acid is replicated. Unless specifically stated,“amplifying” refers to a single replication or to an arithmetic,logarithmic, or exponential amplification.

As used herein, the term “target amplicon” refers to an amplificationproduct having the target sequence of interest and resulting form anamplification reaction, e.g., a polymerase chain reaction (PCR).

As used herein, the terms “extend”, “extension” and “extending” are usedinterchangeably and refer to a process whereby an oligonucleotide isincreased in length at the 3′ end according to a template target nucleicacid sequence. Unless specifically stated, “extend” refers to a singleexpansion or to a plurality of parallel or multiple expansions of atarget or multiple target nucleic acid target sequences.

As used herein, the term “determining a nucleotide base sequence” or theterm “determining information about a sequence” encompasses “sequencedetermination” and also encompasses other levels of information such aseliminating one or more possibilities for a sequence. It is noted thatperforming sequence determination of a polynucleotide typically yieldsequivalent information regarding the sequence of a perfectlycomplementary (100% complementary) polynucleotide and thus is equivalentto sequence determination performed directly on a perfectlycomplementary polynucleotide.

The term “nucleic acid sequence” as used herein can refer to the nucleicacid material itself and is not restricted to the sequence information(i.e. the succession of letters chosen among the five base letters A, C,G, T, or U) that biochemically characterizes a specific nucleic acid,for example, a DNA or RNA molecule. Nucleic acids shown herein arepresented in a 5′→3′ orientation unless otherwise indicated.

The term “mobility-dependent separation” as used herein can refer to theseparation of nucleic acid fragments due to the charge and sizeassociated with the fragment.

The term “fluorescent dye” as used herein refers to moieties that absorblight energy at a defined excitation wavelength and emit light energy ata different wavelength. Preferably the fluorescent dyes selected for useare spectrally resolvable. As used herein, “spectrally resolvable” meansthat the dyes can be distinguished on the basis of their spectralcharacteristics, particularly fluorescence emission wavelength, underconditions of operation. For example, the identity of the one or moreterminal nucleotides can be correlated to a distinct wavelength ofmaximum light emission intensity, or perhaps a ratio of intensities atdifferent wavelengths.

The term “nucleobase” or “base” as used herein refers to anitrogen-containing heterocyclic moiety capable of forming Watson-Cricktype hydrogen bonds with a complementary nucleobase or nucleobaseanalog, e.g. a purine, a 7-deazapurine, or a pyrimidine. Typicalnucleobases are the naturally occurring nucleobases adenine, guanine,cytosine, 5 mC, uracil, thymine, and analogs of naturally occurringnucleobases, e.g. 7-deazaadenine, 7-deaza-8-azaadenine, 7-deazaguanine,7-deaza-8-azaguanine, N6-Δ2 isopentenyl-adenine(6iA),N6-Δ2-isopentenyl-2-methylthioadenine (2ms6iA),N2-dimethyl-guanine(dmG), 7-methylguanine (7mG), inosine, nebularine,nitropyrrole, nitroindole, 2-amino-purine, 2,6-diamino-purine,hypoxanthine, pseudouridine, pseudocytidine, pseudoisocytidine,5-propynyl-cytidine, isocytidine, isoguanine, 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, O⁶-methylguanine,N⁶-methyl-adenine, O⁴-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, 4-methylindole, pyrazolo[3,4-D]pyrimidines (see,e.g., U.S. Pat. Nos. 6,143,877 and 6,127,121 and PCT PublishedApplication WO 01/38584) and ethenoadenine. Nonlimiting examples ofnucleotide bases can be found, e.g., in Fasman, Practical Handbook ofBiochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton,Fla. (1989).

As used herein, the terms “polynucleotide”, “nucleic acid”, or“oligonucleotide” refers to a linear polymer of nucleosides (includingdeoxyribonucleosides, ribonucleosides, or analogs thereof) joined byinter-nucleosidic linkages. Whenever a polynucleotide such as anoligonucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′→3′ orderfrom left to right and that “A” denotes deoxyadenosine, “C” denotesdeoxycytidine, “G” denotes deoxyguanosine, and “T” denotesdeoxythymidine, unless otherwise noted. The letters A, C, G, and T canbe used to refer to the bases themselves, to nucleosides, or tonucleotides comprising the bases, as is standard in the art. Innaturally occurring polynucleotides, the inter-nucleoside linkage istypically a phosphodiester bond, and the subunits are referred to as“nucleotides.” Oligonucleotide primers comprising other inter-nucleosidelinkages, such as phosphorothioate linkages, are used in certainembodiments of the teachings. It will be appreciated that one or more ofthe subunits that make up such an oligonucleotide primer with anon-phosphodiester linkage can not comprise a phosphate group. Suchanalogs of nucleotides are considered to fall within the scope of theterm “nucleotide” as used herein, and nucleic acids comprising one ormore inter-nucleoside linkages that are not phosphodiester linkages arestill referred to as “polynucleotides”, “oligonucleotides”, etc.

As used herein “sequence determination”, “determining a nucleotide basesequence”, “sequencing”, and like terms includes determination ofpartial as well as full sequence information. That is, the term includessequence comparisons, fingerprinting, and like levels of informationabout a target polynucleotide, as well as the express identification andordering of each nucleoside of the target polynucleotide within a regionof interest. In certain embodiments, “sequence determination” comprisesidentifying a single nucleotide, while in other embodiments more thanone nucleotide is identified. Identification of nucleosides,nucleotides, and/or bases are considered equivalent herein. It is notedthat performing sequence determination on a polynucleotide typicallyyields equivalent information regarding the sequence of a perfectlycomplementary polynucleotide and thus is equivalent to sequencedetermination performed directly on a perfectly complementarypolynucleotide.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, primerset(s), etc. in the appropriate containers) and/or supporting materials(e.g., buffers, written instructions for performing the assay etc.) fromone location to another. For example, kits can include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. As used herein, the term “fragmented kit”refers to a delivery system comprising two or more separate containersthat each contain a subportion of the total kit components. Thecontainers may be delivered to the intended recipient together orseparately. For example, a first container may contain an enzyme for usein an assay, while a second container contains oligonucleotides. Indeed,any delivery system comprising two or more separate containers that eachcontains a subportion of the total kit components are included in theterm “fragmented kit.” In contrast, a “combined kit” refers to adelivery system containing all of the components of a reaction assay ina single container (e.g., in a single box housing each of the desiredcomponents). The term “kit” includes both fragmented and combined kits.

As will be appreciated by one of ordinary skill in the art, referencesto templates, oligonucleotides, primers, etc., generally meanpopulations or pools of nucleic acid molecules that are substantiallyidentical within a relevant region rather than single molecules. Forexample, a “template” generally means a plurality of substantiallyidentical template molecules; a “primer” generally means a plurality ofsubstantially identical primer molecules, and the like.

Cycle sequencing involves adding to a target nucleic acid or anamplification product thereof, sequencing primer, deoxynucleotidetriphosphates (dNTPs), dye-labeled chain terminating nucleotides (e.g.,dideoxynucleotide triphosphates (ddNTPs-dyes)), and DNA polymerase,followed by thermal cycle sequencing. Standard cycle sequencingprocedures are well established. Cycle sequencing procedures aredescribed in more detail, for example, in U.S. Pat. No. 5,741,676, andU.S. Pat. No. 5,756,285, each herein incorporated by reference in itsentirety. In certain embodiments, “cycle sequencing” comprises dNTPS, asequencing primer (labeled or not), ddNTPs (labeled or not) and DNApolymerase as known to one of skill in the art. It is noted that alabeled sequencing primer can provide fragment analysis informationand/or determination of the sequence of a target nucleic acid oramplification product thereof.

According to various embodiments of the present teachings, provided is achemically-enhanced primer comprising an oligonucleotide sequence, anegatively charged moiety (NCM) and at least one nuclease-resistantlinkage.

In some embodiments the at least one nuclease-resistant linkage includesbut is not limited to at least one phosphorothioate linkage (PS) or atleast one boronophosphate linkage. In other embodiments thenuclease-resistant linkage is not present in the chemically-enhancedprimer. In yet other embodiments, a chemically-enhanced primer maycomprise an oligonucleotide sequence, a negatively charged moiety (NCM),where the oligonucleotide inter-nucleotide linkages consist ofphosphodiester inter-nucleotide linkages.

The primer can be used to prime a target nucleic acid in a sequencingreaction, herein referred to as a chemically-enhanced sequencing primeror for fragment analysis, herein referred to as a chemically-enhancedextension primer. The oligonucleotide sequence can be a universal primeror a gene specific nucleotide sequence. Examples of universal primersinclude but are not limited to M13 (P/N 402071 and 402072, AppliedBiosystems), US1 (UNISEQ, PLoS Medicine 3(10)e431 (2006)), T7 (P/N402126, but without dye, Applied Biosystems), SP6 (P/N 402128, butwithout dye, Applied Biosystems), and T3 (P/N 402127, but without dye,Applied Biosystems). See the ABI PRIMS® 377 DNA Sequence 96-Lane UpgradeUser's Manual for primer sequences. The oligonucleotide sequence canalso contain a dye-label such as a fluorescent label. In variousembodiments of the present teachings the NCM can be located at theterminal 5′ end of the oligonucleotide sequence or within theoligonucleotide sequence. Examples of NCM include but are not limited toa phosphodiester moiety having a structure of the formula

(which is introduced to the chemically-enhanced primer by reacting aphosphoramidite₇ (available from Glen Research) with an appropriatereaction partner containing an oligonucleotide) referred to here as a(C)_(n) spacer, wherein n can be from 1-12, the amino acids asparticacid and glutamic acid as well as nucleotides and nucleotide analogs(dATP, dCTP, dGTP and dTTP). The NCM can contain only one negativelycharged monomer or a plurality of negatively charged moieties, forexample at least five, ten, 12, 15, 18, 20, 24 or more repeat units ofthe spacer, for example, (Cn)_(x). where x is any integer between 1 andat least 11, at least 12, at least 15, at least 18, at least 20, atleast 24 or 30 Cn spacers where “n” is 3 or 6, e.g., C3 spacers, C6spacers or a combination of C3 and C6 spacers in a linear arrangement ora branched arrangement. The C3 and C6 spacers individually or incombination can also form a branched NCM by forming a doubler or atrebler such as, for example, (C3)₃-treb-M13 or [(C3)₂-treb]-treb-M13,where the NCM is represented by (C3)₃-treb or [(C3)₂-treb]-treb and M13represents the oligonucleotide sequence, as would be known to one ofskill in the art. The NCM can also contain a dye-label such as afluorescent label. In various embodiments at least none, at least one,at least two or more phosphorothioate linkages can be at a terminal 3′end of the oligonucleotide sequence. The presence of at least onenuclease-resistant linkage provides resistance to digestion by 3′-5′nucleases such as Exonuclease I (P/N M0293S New England Biolabs,Ipswich, Mass.), Exo III (P/N MO206S, New England Biolabs, Ipswich,Mass.), Pfu (Promega, P/N M7741, Madison, Wis.), and DNA pol I (P/NM0209S, New England Biolabs, Ipswich, Mass.). The resistance of thechemically-enhanced primer to nuclease digestion offers the advantage ofeliminating a PCR clean-up step in the PCR to sequencing protocol.Removal of the extra non-nuclease resistant amplification primers leftover from the PCR step can be accomplished in the sequencing reactionmixture. A brief exposure of the PCR amplification reaction to thenuclease within the sequencing reaction mixture degrades thenon-nuclease resistant amplification primers followed by an inactivationof the nuclease. The chemically-enhanced primer remains available forthe sequencing reaction while the non-nuclease resistant amplificationprimers and the nuclease have been removed and inactivated,respectively.

In some embodiments the chemically-enhanced primer has a structure ofFormula I:

wherein B is a nucleobase; K is S or O; each n is independently aninteger of 1 to 12; m is 0 or 1; x is an integer of 1 to about 50; z isan integer of 3 to about 100; W is OH, F, OMe, or H; and Nt is a moietyhaving a formula:

For chemically-enhanced primers having a structure of Formula I, OLIGOrepresents the portion of the chemically-enhanced primer of Formula Ithat comprises an oligonucleotide. Each nucleotide of theoligonucleotide comprises a nucleobase B portion and a ribose portion:

The chemically-enhanced primer of Formula I may comprise one or more B,wherein B is a naturally occurring nucleobase. In other embodiments, thechemically-enhanced primer of Formula I may comprise one or more B,wherein B is a nucleobase analog.

The chemically-enhanced primer of Formula I may have only onephosphothiorate linkage, wherein m is 0, having a structure of FormulaI-A:

The chemically-enhanced primer of Formula I may be labeled with a dye,including dyes that are fluorescent. The chemically-enhanced primer ofFormula I may include one or more B labeled with a dye, and isrepresented as B^(f). In some embodiments, the 3′ terminal nucleotide ofthe chemically-enhanced primer has a fluorescently labeled B. Thechemically-enhanced primer may contain a 3′ fluorescently labeledterminal nucleotide wherein the B of the 3′ terminal nucleotide is anucleobase analog. Alternatively, the chemically-enhanced primer maycontain a 5′ terminal nucleotide having a fluorescently labeled B, whichcan be represented as B^(f). In some embodiments, wherein thechemically-enhanced primer contains a 5′ terminal nucleotide containingthe fluorescently labeled nucleobase, B^(f), the labeled nucleobase is anucleobase analog. In other embodiments, the chemically-enhanced primermay contain a fluorescently labeled NCM attached directly or indirectlyto one of a plurality of NCMs and/or a linker moiety to the 5′ terminalnucleotide of the primer. Additionally, the chemically-enhanced primerof Formula I may be fluorescently labeled on the nucleobase of anucleotide located at an internal position of the oligonucleotide, andthe internal fluorescently labeled nucleotide may be selected to be atany position of the non-terminal portion of the oligonucleotide.

When the chemically-enhanced primer of Formula I contains a fluorescentlabel, the chemically-enhanced primer may have a structure of one of thefollowing formulae:

wherein FL is a dye label and B¹ is a dye labeled nucleobase. Fl andB^(f) may each represent a fluorescent dye label.

For the chemically-enhanced primer of Formula I, each n canindependently be an integer of 1 to 12. In some embodiments, n is 1, 2,3, 4, 5, 6, 7, 8, or 9. In some embodiments, n is 3. In otherembodiments, n is 4. Alternatively, n may be 6. In some embodiments ofthe chemically enhanced primer of Formula I, when x is greater than 2, afirst instance of n is selected to be 3 and a second instance of n isselected to be 6. In further embodiments of the chemically-enhancedprimers of Formula I, when x is greater than 2, more than one instanceof n is selected to be 3, and more than one instance of n is selected tobe 6. In yet other embodiments, when x is greater than 5, a plurality ofn is selected to be 3, and a second plurality of n is selected to be 6.

The chemically-enhanced primer of Formula I may have m=1 or m=0. In someembodiments the chemically-enhanced primer of Formula I has m=0.

The chemically-enhanced primer of Formula I may have x, wherein x is aninteger of 1 to about 50. In some of the embodiments of thechemically-enhanced primer of Formula I, x is 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10. In other embodiments, x is 10, 15, 18, 20 or 24. In someembodiments, x is 5, 8, 9, 10 or 15. In other embodiments, x is 11, 12,13, 14, 17 or 20. In other embodiments, x is 30. In further embodiments,x is at least 5, at least 6, at least 8, at least 9, at least 10, atleast 15 at least 18, at least 20, or at least 24. In some embodiments,x is 15. In yet other embodiments, x is 8 or 9.

In some embodiments, the chemically-enhanced primers comprise a secondplurality y of

moieties, wherein y is an integer of 1-20. In some embodiments, when afirst plurality x of n has a value of a first integer, then a secondplurality y of n is an integer of 1 to 20. In some embodiments, thechemically-enhanced primer may have a first plurality of n wherein n is3 and x is 15, and a second plurality of n wherein n is 6 and x is 5.All combinations of n, x and y are contemplated for use in thechemically-enhanced primers of Formula I.

In the chemically-enhanced primer of Formula I, z is an integer of 3 toabout 100. In some embodiments, z is an integer of 5 to 50, 5 to 40, or5 to about 30. In other embodiments, z is an integer of 5 to 25, or 5 to20.

In some of the embodiments of the chemically-enhanced primer of FormulaI, K is S. In other embodiments, K is O.

In some embodiments of the chemically-enhanced primer of Formula I, W isH or OH.

The chemically-enhanced primer of Formula I, I-B, I-C, I-E, I-F, or I-G,may have any combination of B, B^(f), FL, K, m, n, W, x, and z of theranges and selections disclosed above.

The chemically-enhanced primer of Formula I-D may have any combinationof B, FL, K, m, n, W, x, and z of the ranges and selections disclosedabove

The chemically-enhanced primer of Formula I-A, I-H, I-J or I-K, may haveany combination of B, B^(f), FL, K, m, n, W, x, and z of the ranges andselections disclosed above.

In other embodiments, the chemically-enhanced primer is a compoundhaving a structure of Formula II:

wherein B is a nucleobase; K is S or O; each n is independently aninteger of 1 to 12; m is 0 or 1; x is an integer of 1 to about 50; z isan integer of 3 to about 100; W is OH, F, OMe, or H; and Nt is a moietyhaving a formula:

The chemically-enhanced primer of Formula II may be referred to as adoubler, and represents a branched arrangement of NCM moieties.

The chemically-enhanced primer of Formula II may comprise one or more B,wherein B is a naturally occurring nucleobase. In other embodiments, thechemically-enhanced primer of Formula II may comprise one or more B,wherein B is a nucleobase analog.

The chemically-enhanced primer of Formula II may have only onephosphothiorate linkage, wherein m is 0.

The chemically-enhanced primer of Formula II may be labeled with a dye,including dyes that are fluorescent. The chemically-enhanced primer ofFormula II may include one or more B labeled with a dye, and isrepresented as B^(f). In some embodiments, the 3′ terminal nucleotide ofthe chemically-enhanced primer has a fluorescently labeled B. Thechemically-enhanced primer may contain a 3′ fluorescently labeledterminal nucleotide wherein the B of the 3′ terminal nucleotide is anucleobase analog. Alternatively, the chemically-enhanced primer maycontain a 5′ terminal nucleotide having a fluorescently labeled B, whichcan be represented as B^(f). In some embodiments, wherein thechemically-enhanced primer contains a 5′ terminal nucleotide containingthe fluorescently labeled nucleobase, B^(f), the labeled nucleobase is anucleobase analog. In other embodiments, the chemically-enhanced primermay contain a fluorescently labeled NCM attached directly or indirectlyto one of a plurality of NCMs and/or a linker moiety to the 5′ terminalnucleotide of the primer. Additionally, the chemically-enhanced primerof Formula II may be fluorescently labeled on the nucleobase of anucleotide located at an internal position of the oligonucleotide, andthe internal fluorescently labeled nucleotide may be selected to be atany position of the non-terminal portion of the oligonucleotide.

For the chemically-enhanced primer of Formula II, n can be an integer of1 to 9. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. In someembodiments, n is 3. In other embodiments, n is 4. Alternatively, n maybe 6. In some embodiments of the chemically-enhanced primer of FormulaII, when x is greater than 2, a first instance of n is selected to be 3and a second instance of n is selected to be 6. In further embodimentsof the chemically-enhanced primers of Formula I, when x is greater than2, more than one instance of n is selected to be 3, and more than oneinstance of n is selected to be 6. In yet other embodiments, when x isgreater than 5, a plurality of n is selected to be 3, and a secondplurality of n is selected to be 6.

The chemically-enhanced primer of Formula II may have m=1 or m=0. Insome embodiments the chemically-enhanced primer of Formula II has m=0.

The chemically-enhanced primer of Formula II may have x wherein x is aninteger of 1 to about 50. In some of the embodiments of thechemically-enhanced primer of Formula II, x is 1, 2, 3, 4, 5, 6, 7, 8,9, or 10. In other embodiments, x is 10, 15, 18, 20 or 24. In someembodiments, x is 5, 8, 9, 10 or 15. In other embodiments, x is 11, 12,13, 14, 17 or 20. In other embodiments, x is 30. In further embodiments,x is at least 5, at least 6, at least 8, at least 9, at least 10, atleast 15 at least 18, at least 20, or at least 24. In some embodiments,x is 15. In yet other embodiments, x is 8 or 9.

In some embodiments, the chemically-enhanced primers comprise a secondplurality y of

moieties, wherein y is an integer of 1-20. In some embodiments, when afirst plurality x of n has a value of a first integer, then a secondplurality y of n is an integer of 1 to 20. In some embodiments, thechemically-enhanced primer may have a first plurality of n wherein n is3 and x is 15, and a second plurality of n wherein n is 6 and x is 5.All combinations of n, x and y are contemplated for use in thechemically-enhanced primers of Formula II.

In the chemically-enhanced primer of Formula II, z is an integer of 3 toabout 100. In some embodiments, z is an integer of 5 to 50, 5 to 40, or5 to about 30. In other embodiments, z is an integer of 5 to 25, or 5 to20.

In some of the embodiments of the chemically-enhanced primer of FormulaII, K is S. In other embodiments, K is O.

In some embodiments of the chemically-enhanced primer of Formula II, Wis H or OH.

The chemically-enhanced primer of Formula II may have any combination ofB, K, m, n, W, x, and z of the ranges and selections disclosed above.

In yet other embodiments, the chemically-enhanced primer is a compoundhaving a structure of the Formula III:

wherein B is a nucleobase; K is S or O; each n is independently aninteger of 1 to 12; m is 0 or 1; x is an integer of 1 to about 50; z isan integer of 3 to about 100; W is OH, F, OMe, or H; and Nt is a moietyhaving a formula:

The chemically-enhanced primer of Formula III may be referred to as atrebler and represents a branched arrangement of NCM moieties.

The chemically-enhanced primer of Formula III may comprise one or moreB, wherein B is a naturally occurring nucleobase. In other embodiments,the chemically-enhanced primer of Formula III may comprise one or moreB, wherein B is a nucleobase analog.

The chemically-enhanced primer of Formula III may have only onephosphothiorate linkage, wherein m is 0.

The chemically-enhanced primer of Formula III may be labeled with a dye,including dyes that are fluorescent. The chemically-enhanced primer ofFormula III may include one or more B labeled with a dye, and isrepresented as B^(f). In some embodiments, the 3′ terminal nucleotide ofthe chemically-enhanced primer has a fluorescently labeled B. Thechemically-enhanced primer may contain a 3′ fluorescently labeledterminal nucleotide wherein the B of the 3′ terminal nucleotide is anucleobase analog. Alternatively, the chemically-enhanced primer maycontain a 5′ terminal nucleotide having a fluorescently labeled B, whichcan be represented as B^(f). In some embodiments, wherein thechemically-enhanced primer contains a 5′ terminal nucleotide containingthe fluorescently labeled nucleobase, B^(f), the labeled nucleobase is anucleobase analog. In other embodiments, the chemically-enhanced primermay contain a fluorescently labeled NCM attached directly or indirectlyto one of a plurality of NCMs and/or a linker moiety to the 5′ terminalnucleotide of the primer. Additionally, the chemically-enhanced primerof Formula II may be fluorescently labeled on the nucleobase of anucleotide located at an internal position of the oligonucleotide, andthe internal fluorescently labeled nucleotide may be selected to be atany position of the non-terminal portion of the oligonucleotide.

For the chemically-enhanced primer of Formula III, n can be an integerof 1 to 9. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. Insome embodiments, n is 3. In other embodiments, n is 4. Alternatively, nmay be 6. In some embodiments of the chemically-enhanced primer ofFormula III, when x is greater than 2, a first instance of n is selectedto be 3 and a second instance of n is selected to be 6. In furtherembodiments of the chemically-enhanced primers of Formula III, when x isgreater than 2, more than one instance of n is selected to be 3, andmore than one instance of n is selected to be 6. In yet otherembodiments, when x is greater than 5, a plurality of n is selected tobe 3, and a second plurality of n is selected to be 6.

The chemically-enhanced primer of Formula III may have m=1 or m=0. Insome embodiments the chemically-enhanced primer of Formula III has m=0.

The chemically-enhanced primer of Formula III may have x wherein x is aninteger of 1 to about 30. In some of the embodiments of thechemically-enhanced primer of Formula II, x is 1, 2, 3, 4, 5, 6, 7, 8,9, or 10. In other embodiments, x is 10, 15, 18, 20 or 24. In someembodiments, x is 5, 8, 9, 10 or 15. In other embodiments, x is 11, 12,13, 14, 17 or 20. In other embodiments, x is 30. In further embodiments,x is at least 5, at least 6, at least 8, at least 9, at least 10, atleast 15 at least 18, at least 20, or at least 24. In some embodiments,x is 15. In yet other embodiments, x is 8 or 9.

In the chemically-enhanced primer of Formula III, z is an integer of 3to about 100. In some embodiments, z is an integer of 5 to 50, 5 to 40,or 5 to about 30. In other embodiments, z is an integer of 5 to 25, or 5to 20.

In some embodiments, the chemically-enhanced primers comprise a secondplurality y of

moieties, wherein y is an integer of 1-20. In some embodiments, when afirst plurality x of n has a value of a first integer, then a secondplurality y of n is an integer of 1 to 20. In some embodiments, thechemically-enhanced primer may have a first plurality of n wherein n is3 and x is 15, and a second plurality of n wherein n is 6 and x is 5.All combinations of n, x and y are contemplated for use in thechemically-enhanced primers of Formula III.

In some of the embodiments of the chemically-enhanced primer of FormulaIII, K is S. In other embodiments, K is O.

In some embodiments of the chemically-enhanced primer of Formula III, Wis H or OH.

The chemically-enhanced primer of Formula III may have any combinationof B, K, m, n, W, x, and z of the ranges and selections disclosed above.

Other embodiments of the chemically-enhanced primer are represented byFormula IV:

wherein each instance of n is independently an integer of 1 to 12; x isan integer of 1 to 50; v is an integer of 1 to 9; t is 0 or 1; LINKERcomprises 3-100 atoms;OLIGO has a structure of the following formula:

wherein B is a nucleobase; K is S or O; m is 0 or 1; z is an integer of3 to about 100; W is OH, F, OMe, or H; and Nt is a moiety having aformula:

The chemically-enhanced primer of Formula IV, may comprise one or more Bwherein B is a naturally occurring nucleobase. In other embodiments, thechemically-enhanced primer of Formula IV may comprise one or more B,wherein B is a nucleobase analog.

The chemically-enhanced primer of Formula IV may be labeled with a dye,including dyes that are fluorescent. The chemically-enhanced primerhaving a formula of (Cn)_(x)-OLIGO may include one or more B labeledwith a dye, and is represented as B^(f). In some embodiments, when thechemically-enhanced primer has at least one B labeled with a dye, the Bmay be a nucleobase analog. In some embodiments, the 3′ terminalnucleotide of the chemically-enhanced primer has a fluorescently labeledB, which can be represented as B^(f). Alternatively, thechemically-enhanced primer may contain a 5′ terminal nucleotide having afluorescently labeled B, which can be represented as B^(f).Additionally, the chemically-enhanced primer having a formula of(Cn)_(x)-OLIGO, may be fluorescently labeled on the nucleobase of anucleotide located at an internal position of the oligonucleotide, andthe internal fluorescently labeled nucleotide may be selected to be atany position of the non-terminal portion of the oligonucleotide. Inother embodiments, the chemically-enhanced primer may contain afluorescently labeled NCM attached directly or indirectly to one of aplurality of NCM5 and/or to a NCM linker moiety forming a covalentattachment to the 5′ terminal nucleotide of the primer.

LINKER is an NCM linker and may comprise 3-100 atoms and include ether,amide, phosphodiester, and ester moieties to form a covalent linkagebetween the NCM and the oligonucleotide. LINKER may be attached to the5′ carbon of the ribose of the nucleotide at the 5′ terminus of theoligonucleotide. In some embodiments, LINKER is present. In otherembodiments the NCM phosphodiester moiety or moieties are directlyattached to OLIGO.

For the chemically-enhanced primer of Formula IV, v can be an integer of1 to 9. In some embodiments, v is 1. In other embodiments, v is 2. Inyet other embodiments, v is 3.

For the chemically-enhanced primer of Formula IV, n can be an integer of1 to 12. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. Inother embodiments, n is an integer of 1 to 9. In some embodiments, n is3. In other embodiments, n is 4. Alternatively, n may be 6 Thechemically-enhanced primer of Formula IV has x, wherein x is an integerof 1 to about 30. In some of the embodiments of the chemically-enhancedprimer of Formula IV, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In otherembodiments, x is 10, 15, 18, 20 or 24. In some embodiments, x is 5, 8,9, or 15. In other embodiments, x is 11, 12, 13, 14, 17 or 20. Infurther embodiments, x is at least 5, at least 6, at least 8, at least9, at least 10, at least 15 at least 18, at least 20, or at least 24. Insome embodiments, x is 15. In yet other embodiments, x is 8 or 9.

In some embodiments of the chemically-enhanced primer of Formula IV,when x is greater than 2, a first instance of n is selected to be 3 anda second instance of n is selected to be 6. In further embodiments ofthe chemically-enhanced primers of Formula IV, when x is greater than 2,more than one instance of n is selected to be 3, and more than oneinstance of n is selected to be 6. In yet other embodiments, when x isgreater than 5, a plurality of n is selected to be 3, and a secondplurality of n is selected to be 6.

In some embodiments, the chemically-enhanced primers comprise a secondplurality y of

moieties, wherein y is an integer of 1-20. In some embodiments, when afirst plurality x of n has a value of a first integer, then a secondplurality y of n is an integer of 1 to 20. In some embodiments, thechemically-enhanced primer may have a first plurality of n wherein n is3 and x is 15, and a second plurality of n wherein n is 6 and x is 5.All combinations of n, x and y are contemplated for use in thechemically-enhanced primers of Formula IV.

The chemically-enhanced primer having a formula of Formula IV has m=1 orm=0. In some embodiments the chemically-enhanced primer of Formula IVhas m=0.

The chemically-enhanced primer having a formula of Formula IV has z,wherein z is an integer of 3 to about 100. In some embodiments, z is aninteger of 5 to 50, 5 to about 40, or 5 to about 30. In otherembodiments, z is an integer of 5 to 25, or 5 to 20.

In some of the embodiments of the chemically-enhanced primer of FormulaIV, K is S. In other embodiments, K is O.

In some embodiments of the chemically-enhanced primer of Formula IV, Wis H or OH.

The chemically-enhanced primer of Formula IV, may have any combinationof B, n, t, v, x, m, y, z, K or W of the ranges and selections disclosedabove.

In some embodiments, the chemically-enhanced primer of Formula IV is achemically-enhanced primer (Cn)_(x)-OLIGO, wherein (Cn)_(x) has astructure of the following formula:

wherein each instance of n is independently an integer of 1 to 12; and xis an integer of 1 to about 30; and OLIGO has a structure of thefollowing formula:

wherein B, K, m, z, y, Nt, and W are as defined above for Formula IV.

For the chemically-enhanced primer having a formula of (Cn)_(x)-OLIGO, vis 1, t is 0, no LINKER is present, and the chain of NCM moieties areattached to OLIGO directly.

The chemically-enhanced primer having a formula of (Cn)_(x)-OLIGO, mayhave any combination of B, n, x, m, z, K or W of the ranges andselections disclosed above for Formula IV.

In yet other embodiments, the chemically-enhanced primer is representedby the following formulae:

(Cn)_(x)-OLIGO*, wherein (Cn)_(x) has a structure of the followingformula:

wherein each instance of n is independently an integer of 1 to 12; and xis an integer of 1 to about 30; OLIGO* has a structure of the followingformula:

wherein B, K, m, z, y, Nt, and W are as defined above for Formula IV.

For the chemically-enhanced primer having a formula of (Cn)_(x)-OLIGO*,v is 1, t is 0, no LINKER is present, and the chain of NCM moieties areattached to OLIGO* directly.

The chemically-enhanced primer having a formula of (Cn)_(x)-OLIGO*, mayhave any combination of B, n, x, m, z, or W of the ranges and selectionsdisclosed above for Formula IV.

Chemically-enhanced primers having a formula of (Cn)_(x) Formula VI-A1include, but are not limited to:

(Cn)_(x)-US1, where n is 1 to 9 and x is 1 to about 30. In someembodiments, (Cn)_(x)-US1 is (C3)₁-US1, (C3)₂-US1, (C3)₃-US1, (C3)₄-US1,(C3)₅-US1, (C3)₆-US1, (C3)₇-US1, (C3)₈-US1, (C3)₉-US1, (C3)₁₀-US1,(C3)₁₁-US1, (C3)₁₂-US1, (C3)₁₃-US1, (C3)₁₄-US1, (C3)₁₅-US1, (C3)₁₆-US1,(C3)₁₇-US1, (C3)₁₈-US1, (C3)₂₁-US1, (C3)₂₄-US1, (C3)₂₇-US1, or(C3)₃₀-US1. In some embodiments, (Cn)_(x)-US1 is a forward primer andmay have any x as described above. In other embodiments, (Cn)_(x)-US1 isa reverse primer and may have any x as described above.

(Cn)_(x)-M13-forward, where n is 1 to 9 and x is 1 to about 30. In someembodiments, (Cn)_(x)-M13-forward is (C3)₁-M13-forward,(C3)₂-M13-forward, (C3)₃-M13-forward, (C3)₄-M13-forward,(C3)₅-M13-forward, (C3)₆-M13-forward, (C3)₇-M13-forward,(C3)₈-M13-forward, (C3)₉-M13-forward, (C3)₁₀-M13-forward,(C3)₁₁-M13-forward, (C3)₁₂-M13-forward, (C3)₁₃-M13-forward,(C3)₁₄-M13-forward, (C3)₁₅-M13-forward, (C3)₁₆-M13-forward,(C3)₁₇-M13-forward, (C3)₁₈-M13-forward, (C3)₂₁-M13-forward,(C3)₂₄-M13-forward, (C3)₂₇-M13-forward, or (C3)₃₀-M13-forward.

(Cn)_(x)-M13-reverse, where n is 1 to 9 and x is 1 to about 30. In someembodiments, (Cn)_(x)-M13-reverse is (C3)₁-M13-reverse,(C3)₂-M13-reverse, (C3)₃-M13-reverse, (C3)₄-M13-reverse,(C3)₅-M13-reverse, (C3)₆-M13-reverse, (C3)₇-M13-reverse,(C3)₈-M13-reverse, (C3)₉-M13-reverse, (C3)₁₀-M13-reverse,(C3)₁₁-M13-reverse, (C3)₁₂-M13-reverse, (C3)₁₃-M13-reverse,(C3)₁₄-M13-reverse, (C3)₁₅-M13-reverse, (C3)₁₆-M13-reverse,(C3)₁₇-M13-reverse, (C3)₁₈-M13-reverse, (C3)₂₁-M13-reverse,(C3)₂₄-M13-reverse, (C3)₂₇-M13-reverse, or (C3)₃₀-M13-reverse.

(Cn)_(x)-T7, where n is 1 to 9 and x is 1 to about 30. In someembodiments, (Cn)_(x)-T7 is (C3)₁-T7, (C3)₂-T7, (C3)₃-T7 (C3)₄-T7,(C3)₅-T7, (C3)₆-T7, (C3)₇-T7, (C3)₈-T7, (C3)₉-T7, (C3)₁₀-T7, (C3)₁₁-T7,(C3)₁₂-T7, (C3)₁₃-T7, (C3)₁₄-T7, (C3)₁₅-T7, (C3)₁₆-T7, (C3)₁₇-T7,(C3)₁₈-T7, (C3)₂₁-T7, (C3)₂₄-T7, (C3)₂₇-T7, or (C3)₃₀-T7. In someembodiments, (Cn)_(x)-T7 is a forward primer and may have any x asdescribed above. In other embodiments, (Cn)_(x)-T7 is a reverse primerand may have any x as described above.

(Cn)_(x)-SP6 where n is 1 to 9 and x is 1 to about 30. In someembodiments, (Cn)_(x)-SP6 is (C3)₁-SP6, (C3)₂-SP6, (C3)₃-SP6, (C3)₄-SP6,(C3)₅-SP6, (C3)₆-SP6, (C3)₇-SP6, (C3)₈-SP6, (C3)₉-SP6, (C3)₁₀-SP6,(C3)₁₁-SP6, (C3)₁₂-SP6, (C3)₁₃-SP6, (C3)₁₄-SP6, (C3)₁₅-SP6, (C3)₁₆-SP6,(C³)₁₇-SP6, (C3)₁₈-SP6, (C3)₂₁-SP6, (C3)₂₄-SP6, (C3)₂₇-SP6, or(C3)₃₀-SP6. In some embodiments, (Cn)_(x)-SP6 is a forward primer andmay have any x as described above. In other embodiments, (Cn)_(x)-SP6 isa reverse primer and may have any x as described above.

(Cn)_(x)-T3 where n is 1 to 9 and x is 1 to about 30. In someembodiments, (Cn)_(x) T3 is (C3)₁-T3, (C3)₂-T3, (C3)₃-T3, (C3)₄-T3,(C3)₅-T3, (C3)₆-T3, (C3)₇-T3, (C3)₈-T3, (C3)₉-T3, (C3)₁₀-T3, (C3)₁₁-T3,(C3)₁₂-T3, (C3)₁₃-T3, (C3)₁₄-T3, (C3)₁₅-T3, (C3)₁₆-T3, (C3)₁₇-T3,(C3)₁₈-T3, (C3)₂₁-T3, (C3)₂₄-T3, (C3)₂₇-T3, or (C3)₃₀-T3. In someembodiments, (Cn)_(x)-T3 is a forward primer and may have any x asdescribed above. In other embodiments, (Cn)_(x)-T3 is a reverse primerand may have any x as described above.

(Cn)_(x)-GSO, where n is 1 to 9, x is 1 to about 30, and GSO is a genespecific oligonucleotide sequence, wherein the gene specificoligonucleotide comprises 50 or fewer nucleotides. In some embodiments,(Cn)_(x) GSO is (C3)₁-GSO, (C3)₂-GSO, (C3)₃-GSO, (C3)₄-GSO, (C3)₅-GSO,(C3)₆-GSO, (C3)₇-GSO, (C3)₈-GSO, (C3)₉-GSO, (C3)₁₀-GSO, (C3)₁₁-GSO,(C3)₁₂-GSO, (C3)₁₃-GSO, (C3)₁₄-GSO, (C3)₁₅-GSO, (C3)₁₆-GSO, (C3)₁₇-GSO,(C3)₁₈-GSO, (C3)₂₁-GSO, (C3)₂₄-GSO, (C3)₂₇-GSO, or (C3)₃₀-GSO. In someembodiments, (Cn)_(x)-GSO is a forward primer and may have any x asdescribed above. In other embodiments, (Cn)_(x)-GSO is a reverse primerand may have any x as described above.

Chemically-enhanced primers having a formula of (Cn)_(x)-OLIGO* include,but are not limited to:

(cn)_(x)-US1* where n is 1 to 9 and x is 1 to about 30. In someembodiments, (Cn)_(x)-US1* is (C3)₁-US1*, (C3)₂-US1*, (C3)₃-US1*,(C3)₄-US1*, (C3)₅-US1*, (C3)₆-US1*, (C3)₇-US1*, (C3)₈-US1*, (C3)₉-US1*,(C3)₁₀-US1*, (C3)₁₁-US1*, (C3)₁₂-US1*, (C3)₁₃-US1*, (C3)₁₄-US1v,(C3)₁₅-US1*, (C3)₁₆-US1*, (C3)₁₇-US1*, (C3)₁₈-US1*, (C3)₂₁-US1*,(C3)₂₄-US1*, (C3)₂₇-US1*, or (C3)₃₀-US1*. In some embodiments,(Cn)_(x)-US1* is a forward primer and may have any x as described above.In other embodiments, (Cn)_(x)-US1* is a reverse primer and may have anyx as described above.

(Cn)_(x)-M13*-forward, where n is 1 to 9 and x is 1 to about 30. In someembodiments, (Cn)_(x)-M13*-forward is (C3)₁-M13*-forward,(C3)₂-M13*-forward, (C3)₃-M13*-forward, (C3)₄-M13*-forward,(C3)₅-M13*-forward, (C3)₆-M13*-forward, (C3)₇-M13*-forward,(C3)₃-M13*-forward, (C3)₉-M13*-forward, (C3)₁₀-M13*-forward,(C3)₁₁-M13*-forward, (C3)₁₂-M13*-forward, (C3)₁₃-M13*-forward,(C3)₁₄-M13*-forward, (C3)₁₅-M13*-forward, (C3)₁₆-M13*-forward,(C3)₁₇-M13*-forward, (C3)₁₃-M13*-forward, (C3)₂₁-M13*-forward,(C3)₂₄-M13*-forward, (C3)₂₇-M13*-forward, or (C3)₃₀-M13*-forward.

(Cn)_(x)-M13*-reverse, where n is 1 to 9 and x is 1 to about 30. In someembodiments, (Cn)_(x)-M13*-reverse is (C3)₁-M13*-reverse,(C3)₂-M13*-reverse, (C3)₃-M13*-reverse, (C3)₄-M13*-reverse,(C3)₅-M13*-reverse, (C3)₆-M13*-reverse, (C3)₇-M13*-reverse,(C3)₃-M13*-reverse, (C3)₉-M13*-reverse, (C3)₁₀-M13*-reverse,(C3)₁₁-M13*-reverse, (C3)₁₂-M13*-reverse, (C3)₁₃-M13*-reverse,(C3)₁₄-M13*-reverse, (C3)₁₅-M13*-reverse, (C3)₁₆-M13*-reverse,(C3)₁₇-M13*-reverse, (C3)₁₃-M13*-reverse, (C3)₂₁-M13*-reverse,(C3)₂₄-M13*-reverse, (C3)₂₇-M13*-reverse, or (C3)₃₀-M13*-reverse.

(Cn)_(x)-T7*, where n is 1 to 9 and x is 1 to about 30. In someembodiments, (Cn)_(x)-T7* is (C3)₁-T7*, (C3)₂-T7*, (C3)₃-T7*, (C3)₄-T7*,(C3)₅-T7*, (C3)₆-T7*, (C3)₇-T7*, (C3)₃-T7*, (C3)₉-T7*, (C3)₁₀-T7*,(C3)₁₁-T7*, (C3)₁₂-T7*, (C3)₁₃-T7*, (C3)₁₄-T7*, (C3)₁₅-T7*, (C3)₁₆-T7*,(C3)₁₇-T7*, (C3)₁₃-T7*, (C3)₂₁-T7*, (C3)₂₄-T7*, (C3)₂₇-T7*, or(C3)₃₀-T7*. In some embodiments, (Cn)_(x)-T7* is a forward primer andmay have any x as described above. In other embodiments, (Cn)_(x)-T7* isa reverse primer and may have any x as described above.

(Cn)_(x)-SP6*, where n is 1 to 9 and x is 1 to about 30. In someembodiments, (Cn)_(x)-SP6* is (C3)₁-SP6*, (C3)₂-SP6*, (C3)₃-SP6*,(C3)₄-SP6*, (C3)₅-SP6*, (C3)₆-SP6*, (C3)₇-SP6*, (C3)₃—SP6*, (C3)₉-SP6*,(C3)₁₀-SP6*, (C3)₁₁-SP6*, (C3)₁₂-SP6*, (C3)₁₃-SP6*, (C3)₁₄-SP6*,(C3)₁₅-SP6*, (C3)₁₆-SP6*, (C3)₁₇-SP6*, (C3)₁₃-SP6*, (C3)₂₁-SP6*,(C3)₂₄-SP6*, (C3)₂₇-SP6*, or (C3)₃₀-SP6*. In some embodiments,(Cn)_(x)-SP6* is a forward primer and may have any x as described above.In other embodiments, (Cn)_(x)-SP6* is a reverse primer and may have anyx as described above.

(Cn)_(x)-GSO*, where n is 1 to 9, and x is 1 to about 30. In someembodiments, (Cn)_(x) T3* is (C3)₁-T3*, (C3)₂-T3*, (C3)₃-T3*, (C3)₄-T3*,(C3)₅-T3*, (C3)₆-T3*, (C3)₇-T3*, (C3)₃-T3*, (C3)₉-T3*, (C3)₁₀-T3*,(C3)₁₁-T3*, (C3)₁₂-T3*, (C3)₁₃-T3*, (C3)₁₄-T3*, (C3)₁₅-T3*, (C3)₁₆-T3*,(C3)₁₇-T3*, (C3)₁₃-T3*, (C3)₂₁-T3*, (C3)₂₄-T3*, (C3)₂₇-T3*, or(C3)₃₀-T3*. In some embodiments, (Cn)_(x)-T3* is a forward primer andmay have any x as described above. In other embodiments, (Cn)_(x)-T3* isa reverse primer and may have any x as described above.

(Cn)_(x)-GSO*, where n is 1 to 9, x is 1 to about 30, and GSO* is a genespecific oligonucleotide sequence, wherein the gene specificoligonucleotide comprises 50 or fewer nucleotides. In some embodiments,(Cn)_(x) GSO* is (C3)₁-GSO*, (C3)₂-GSO*, (C3)₃-GSO*, (C3)₄-GSO*,(C3)₅-GSO*, (C3)₆-GSO*, (C3)₇-GSO*, (C3)₃-GSO*, (C3)₉-GSO*, (C3)₁₀-GSO*,(C³)₁₁-GSO*, (C3)₁₂-GSO*, (C3)₁₃-GSO*, (C3)₁₄-GSO*, (C3)₁₅-GSO*,(C3)₁₆-GSO*, (C3)₁₇-GSO*, (C3)₁₈-GSO*, (C3)₂₁-GSO*, (C3)₂₄-GSO*,(C3)₂₇-GSO*, or (C3)₃₀-GSO*. In some embodiments, (Cn)_(x)-GSO* is aforward primer and may have any x as described above. In otherembodiments, (Cn)_(x)-GSO* is a reverse primer and may have any x asdescribed above.

In further embodiments, the chemically-enhanced primer of Formula IV maybe a doubler, and has a structure of Formula V:

wherein t is 1; LINKER is present; v is 2; and OLIGO, x, and n are asdefined above for Formula IV.

One example of a chemically-enhanced primer of Formula V has a structureof Formula V-A:

wherein n, x, and OLIGO are as defined above for Formula IV, and LINKERhas a structure of the following formula:

The chemically-enhanced primer of Formula V-A may have any combinationof B, n, x, m, y, z, K or W of the ranges and selections disclosed abovefor Formula IV.

In yet another embodiment, the chemically-enhanced primer of Formula IVmay be a trebler and have a structure of Formula VI:

wherein t is 1; LINKER is present; v is 3; and OLIGO, x, and n are asdefined above for Formula IV. One group of chemically-enhanced primersof Formula VI have structures of Formula VI-A:

wherein r is an integer of 2 to 9; and n, x, and OLIGO are as definedfor Formula IV.

For chemically-enhanced primers of Formula VI-A, LINKER has a structureof the following formula:

In the group of chemically-enhanced primers of Formula VI-A, oneexemplary sub-group has a structure of Formula VI-A1:

wherein n, x and OLIGO are as defined for Formula IV.

The chemically-enhanced primer of Formula VI-A may have any combinationof B, n, r, x, m, y, z, K or W of the ranges and selections disclosedabove for Formula IV, Formula VI, and Formula VI-A.

Using the chemically-enhanced sequencing primer, it has been found thathigh quality and highly accurate nucleic acid sequence results can beobtained in about 50% less time overall for a PCR to sequencing resultsworkflow using POP7™ polymer on any of the 3130, 3730 or 3500 capillaryelectrophoresis platforms (Applied Biosystems, Foster City, Calif.) andwith results comparable to the slower POP6™ polymer (Compare FIGS. 1Aand 1B). Specifically, improvement of 5′ sequence resolution to base 1from the chemically-enhanced sequencing primer. Prior to the presentteachings a standard sequencing primer using the BigDye® Terminator Kitv3.1 (Applied Biosystems) with POP-7™ polymer did not providedinterpretable data the first 20-30 bases after the sequencing primerwhile using the POP-6™ polymer and BigDye® Terminator Kit v1.1 was ableto obtain readable sequence by capillary electrophoresis within fivebases of the sequencing primer, yet the throughput time with POP-6polymer is slower (data not shown). The current teachings provide a NCMsequencing primer that provided high quality bases right after thesequencing primer with POP-7™ polymer by capillary electrophoresis andwith resolution typically equivalent or superior in quality to BigDye®Terminator v1.1 with POP-6™ polymer (see FIG. 3 of U.S. Ser. No.61/407,899, filed Oct. 28, 2011 and U.S. Ser. No. 61/408,553, filed Oct.29, 2011). The chemically-enhanced sequencing primer is able to run withPOP-7 polymer having an electrophoretic run time as short as 65 minutesto generate 700 high quality bases starting from the first base using a3500 Genetic Analyzer (Applied Biosystems) (FIG. 1B). In contrast, ittook 135 minutes with POP-6 polymer to produce only 600 high qualitybases (FIG. 1A). The primer of the present teachings in conjunction withthe POP-7 polymer provided a 52% throughput increase compared to theelectrophoresis time with the POP-6 polymer. The throughput wasincreased as well as reducing hands-on-time by eliminating a separatePCR clean-up step prior to initiation of the sequencing reaction. (FIG.1A and FIG. 1B). Overall, it can be seen that amplification, PCRclean-up and sequencing detection steps can each provide savings in runtime, using differing aspects of the chemically-enhanced primers. Thenuclease resistant aspect of the chemically-enhanced primers may providereduced amplification and PCR clean-up time requirements, and the NCMaspect of the primers may allow efficacious separation in shorter runtimes, than standard sequencing primers can provide.

As shown in FIGS. 13-16 (presented in U.S. Ser. No. 61/407,899, filedOct. 28, 2011 and U.S. Ser. No. 61/408,553, filed Oct. 29, 2011), thechemically-enhanced sequencing primer and workflow was used toinvestigate polymorphisms in Human Leukocyte Antigens (HLA). The HLAsare used for tissue and organ typing as well as tissue and organcross-matching for transplantation matching and evaluation of rejection.The SeCore® HLA-DRB1 (Invitrogen, Carlsbad, Calif.) primer set and groupspecific sequencing primers (GSSP) were used on 34 DNA samples.Sequencing reactions were performed with traditional sequencing primersand with the chemically-enhanced sequencing primers. The sequencingreaction products were electrophoressed on an Applied Biosystems 3500xl™Genetic Analyzer using POP7 polymer (Applied Biosystems). Comparison of5′ resolution and basecalling accuracy and quality was made between thetwo primers. On average the traditional sequencing primers with POP7polymer yielded high quality readable bases by 25 bases after thesequencing primer while the chemically-enhanced sequencing primersyielded high quality bases by base 5 and by base 1 in many cases andalso resulted in increased basecalling accuracy and a 40% decrease inoverall workflow time. Table 1 provides examples of the improvedsequencing quality obtained with the chemically-enhanced primers.

TABLE 1 # of edits # of edits Sample ID allele 1 DNA allele 2 DNABigDye ® Direct Secore 1 D062 DRB1*030101 DRB1*0404 0 0 2 D075DRB1*030101 DRB1*100101 0 0 3 D111 DRB1*010101 DRB1*080101 0 0 4 D115DRB1*030101 DRB1*1503 2 0 5 D125 DRB1*010101 DRB1*070101 0 0 6 D140DRB1*070101 DRB1*1311 0 0 7 D165 DRB1*110101 DRB1*1504 0 2 8 D205DRB1*010201 DRB1*1202 0 0 9 D218 DRB1*1001 DRB1*1320 1 0 1 D099DQB1*030101 DQB1*050101 4 8 2 D108 DQB1*050101 DQB1*0202 0 0 3 D113DQB1*0201 DQB1*050101 2 1 4 D116 DQB1*050301 DQB1*060101 0 0 5 D130DQB1*030302 DQB1*0502 2 2 6 D130 DQB1*030302 DQB1*0502 0 0 7 D135DQB1*0202 DQB1*030101 0 0 8 D150 DQB1*050201 DQB1*030201 1 0 9 D154DQB1*040102 DQB1*060101 0 1 10 D161 DQB1*0201 DQB1*0302 0 0 11 D168DQB1*030101 DQB1*050101 2 3 12 D181 DQB1*0301 DQB1*0501 2 3 13 F2150DQB1*0401/02 DQB1*060101 . . . 0 0 14 F2160 DQB1*0401/02 DQB1*060101-170 1 15 F2297 DQB1*020101-4 . . . DQB1*060101 0 2 16 U415 DQB1*0402DQB1*0601 0 0 17 U415 DQB1*0402 DQB1*0601 1 1 1 D049 DPB1*010101DPB1*040101 0 0 2 D105 DPB1*110101 DPB1*1701 0 0 3 D149 DPB1*1001DPB1*200101 0 0 4 D157 DPB1*010101 DPB1*040101 0 0 5 D161 DPB1*040101DPB1*0601 0 0 6 D164 DPB1*020102 DPB1*0601 0 0 7 D219 DPB1*040101DPB1*0501 0 0 8 U514 DPB1*0201 DPB1*0401 0 0 total edits 17 24

The results in Table 1 illustrate the relative basecalling accuracybetween the SeCore® HLA Sequence and the chemically-enhanced sequencingprimer when sequenced in both directions of exon 2 from HLA-DRB1, DQB1and DPB1 in 34 different samples. uTYPE® HLA Sequencing Software(Invitrogen) aligned the forward and reverse traces to a referencesequence and a HLA library. Basecalling accuracy was assessed by howmany base positions required manual edits to resolve discrepanciesbetween the forward, reverse and reference sequence. Thechemically-enhanced sequencing primer equaled or slightly outperformedthe existing method by requiring fewer manual edits (17) and provided asimplified workflow and faster electrophoresis time vs. 24 edits withthe traditional primers, method and workflow. The chemically-enhancedsequencing primer also improved 5′ mobility seen as 5′ C/A and A/G inDPB1, resolved shoulder problems in HLA-DQB1, and reduced C nucleotidecompression routinely observed in HLA-DRB-1 sequences to substantiallyimprove primary mixed basecalling (data not shown). Thechemically-enhanced sequencing primer and improved workflow improvedpolymorphism detection and more efficient use of allele specificsequencing primers for heterozygous ambiguity resolution resulting insignificant time savings for obtaining data that was superior in qualityto existing methods.

According to various embodiments of the present teachings, a compositionfor sequencing nucleic acid can comprise a polymerase, a nuclease, achemically-enhanced sequencing primer, dNTPs, and a chain terminator(e.g., ddNTPs). In some embodiments, the composition for sequencingnucleic acids can further comprise more than one chemically-enhancedsequencing primer. In some embodiments, the polymerase can comprise Taqpolymerase, for example AmpliTaq Gold polymerase. In some embodiments,the nuclease can comprise exonuclease I. In some embodiments, thechemically-enhanced sequencing primer can comprise at least onephosphorothioate linkage. In other embodiments, the chemically-enhancedsequencing primer can comprise a terminal 3′ end phosphorothioatelinkage. In some embodiments, the ddNTPs can comprise ddNTPs-dyes, forexample fluorescent dye-labeled ddNTPs. In some embodiments thechemically-enhanced sequencing primer can comprise a dye, for example afluorescent dye-labeled oliogonucleotide and/or at least onefluorescently dye-labeled NCM moiety within the NCM compound.

According to various embodiments, the composition for sequencing nucleicacid can comprise a polymerase, for example a DNA polymerase, in anamount of from about 0.01 Unit to about 20 Units, for example, fromabout 0.1 Unit to about 1.0 Unit, or about 0.8 Unit. The composition cancomprise polymerase in an amount within a range having an upper limit offrom about 10 Units to about 20 Units and a lower limit of from about0.01 Unit to about 0.05 Unit. According to various embodiments, thecomposition can comprise a nuclease, for example exonuclease I, in anamount of from about 1 Unit to about 40 Units, for example, from about 2Units to about 15 Units, or about 10 Units. The composition can comprisenuclease in an amount within a range having an upper limit of from about10 Units to about 40 Units, and a lower limit of from about 1 Unit toabout 2 Units.

According to various embodiments, the composition for sequencing nucleicacid can comprise a chemically-enhanced sequencing primer, in an amountof from about 0.1 μM to about 20 μM, for example about 1.0 μM. Thecomposition can comprise a chemically-enhanced sequencing primer in anamount within a range having an upper limit of from about 10 μM to about20 μM and a lower limit of from about 0.05 μM to about 0.1 μM. Accordingto various embodiments, the composition can comprise dNTPs in an amountof from about 20 μM to about 5000 μM, for example, about 500 μM. Thecomposition can comprise dNTPs in an amount within a range having anupper limit of from about 2000 μM to about 5000 μM and a lower limit offrom about 20 μM to about 50 μM. According to various embodiments, thecomposition can comprise ddNTPs in an amount of from about 0.03 μM toabout 10 μM, for example about 3 μM. The composition can comprise ddNTPsin an amount within a range having an upper limit of from about 5 μM toabout 10 μM and a lower limit of from about 0.01 μM to about 0.05 μM.All molar amounts are based on final concentrations of the final volume.

According to various embodiments, the composition can comprise anon-nuclease-resistant amplification primer in an amount of from about0.1 μM to about 20 μM, for example about 1.0 μM. The composition cancomprise a non-nuclease-resistant amplification primer in an amountwithin a range having an upper limit of from about 10 μM to about 20 μMand a lower limit of from about 0.05 μM to about 0.1 μM. All molaramounts are based on final concentrations of the final volume.

According to various embodiments, the composition for sequencing nucleicacid can further comprise a PCR amplification product. In someembodiments, the PCR amplification product can comprise an amplified DNAtarget sequence. In some embodiments, the PCR amplification product cancomprise non-nuclease-resistant amplification primer(s). Thenon-nuclease-resistant amplification primer can comprise, for example,phosphodiester linkages that are sensitive to degradation byexonuclease. In some embodiments, the PCR amplification product cancomprise a target specific amplicon that incorporates nucleic acidsequence capable of annealing to a universal primer.

According to various embodiments of the present teachings, a method ofpreparing a nucleic acid for sequencing can comprise a step ofamplifying the nucleic acid under conditions to produce amplificationreaction products. The nucleic acid can be amplified using, for example,polymerase chain reaction (PCR). The nucleic acid can also be amplifiedusing other methods such as, for example, multiple strand displacementamplification, helicase displacement amplification, nick translation, Qbeta replicase amplification, rolling circle amplification, and otherisothermal amplification methods.

According to various embodiments, the nucleic acid to be amplified cancomprise, for example, RNA, DNA, cDNA, genomic DNA, viral DNA, plasmidDNA, recombinant DNA, amplicon DNA, synthetic DNA or the like. Templatemolecules can be obtained from any of a variety of sources. For example,DNA as a template molecule can be isolated from a sample, which can beobtained or derived from a subject. The word “sample” is used in a broadsense to denote any source of a template on which sequence determinationis to be performed. The phrase “derived from” is used to indicate that asample and/or nucleic acids in a sample obtained directly from a subjectcomprising nucleic acid can be further processed to obtain templatemolecules.

The source of a sample can be of any viral, prokaryotic,archaebacterial, or eukaryotic species or a synthetic species. Incertain embodiments the source can be a human. The sample can comprise,for example, embryonic, cultured cells, tissues or organs, bone, tooth,organ, tissue, preserved, e.g., formalin-fixed paraffin embed (PFPE)organ or tissue, degraded, mummified, or tissue including blood oranother body fluid containing cells, such as sperm, a biopsy sample, orthe like. Mixtures of nucleic acids from different samples and/orsubjects can be combined. Samples can be processed in any of a varietyof ways. Nucleic acids can be isolated, purified, and/or amplified froma sample using known methods.

Amplifying nucleic acid can typically result in a reaction product thatcomprises excess amplification primer and an amplicon (also referred toas an amplification product) that comprises a target nucleic acid.According to various embodiments, a method of preparing nucleic acid forsequencing can comprise removing excess amplification primer from thereaction product. In some embodiments, the amplification primer can beremoved, for example, by adding a nuclease enzyme and providingappropriate conditions for the nuclease to degrade the amplificationprimer. In some embodiments, the amplification primer can be removed bycontacting the amplification reaction product with a reaction mixturecomprising a nuclease enzyme. Nucleases suitable for use in the subjectmethods preferentially degrade single-stranded polynucleotides overdouble-stranded polynucleotides, thus destroying excess primers whileleaving intact double-stranded amplicons available for sequencing insubsequent steps. In various embodiments, the nuclease enzyme cancomprise, for example, exonuclease I. Exonuclease I can be obtained fromvarious commercial suppliers, for example from USB Corp., Cleveland,Ohio. Appropriate reaction conditions can include, for example, optimaltime, temperature, and buffer parameters to provide for nuclease enzymeactivity. In some embodiments, for example, excess amplification primercan be degraded by adding exonuclease I to the amplification reactionproduct and incubating at about 37° C. for about 10 to about 30 min.Exonuclease I can hydrolyze single-stranded DNA in a 3′→5′ direction.

According to various embodiments of a method for preparing a nucleicacid, a reaction mixture can further comprise a chemically-enhancedsequencing primer. The chemically-enhanced sequencing primer can beessentially non-degraded by a reaction mixture comprising a nuclease,for example, exonuclease I, under reaction conditions at which excessamplification primer can be degraded by the nuclease. By “essentiallynon-degraded” it is intended that any degradation that takes place ofthe chemically-enhanced sequencing primer is not of a level thatsignificantly interferes with the process employed to generatesequencing and/or fragment analysis data in the subsequent sequencingreactions or fragment analysis reactions.

According to various embodiments, the chemically-enhanced sequencingprimer can comprise an oligonucleotide sequence. In some embodiments,the chemically-enhanced sequencing primer can comprise one of morenuclease-resistant internucleotide linkage(s). For example, theinternucleotide linkage may be a phosphorothioate linkage. In someembodiments, the chemically-enhanced sequencing primer can comprise anuclease-resistant internucleotide linkage at a terminal 3′ end, at aterminal 5′ end, and/or at one or more internal linkage sites. In someembodiments, the nuclease resistant internucleotide linkage is at leastone phosphorothioate linkage. Chemically-enhanced sequencing primerswere synthesized having one or two phosphorothioate linkages on theterminal 3′ end to protect the chemically-enhanced sequencing primersfrom exonuclease I digestion. The Sp stereoisomer can protect the primerfrom exonuclease I digestion but the Rp steroisomer was found to provideno protection from exonuclease I digestion (data not shown).

The use of a chemically-enhanced sequencing primer having theconfiguration (C3)₈-trebler-M13Rev with either one or twophosphorothioate linkages was evaluated in the sequencing of twotemplates, #30 and #122. It was found that in both templates the firstbase (A) is split into two peaks with the primer having onephosphorothioate linkage, and the first two bases (A and T) were splitwith the primer having two phosphorothioate linkages because thestereoisomers have slightly different mobilities. For the primer withone phosphorothioate linkage, there is either oligo-Sp or oligo-Rp. Inthe case of two phosphorothioate linkages on the terminal 3′ end of theprimer, there were four possible isomers (oligo-Sp-Sp, oligo-Rp-Sp,oligo-Rp-Rp and oligo-Sp-Rp) on the primer with two phosphorothioatelinkages. It was found that as with plasmid sequencing, mobilitydifferences were caused by the Sp and Rp stereoisomers resulted in thesplitting of the first two peaks.

In some embodiments, the chemically-enhanced primer can comprise anegatively charged group/compound/molecule (NCM). For example, the NCMcan be located at the terminal 5′ end of the oligonucleotide sequence orwithin the oligonucleotide sequence. Examples of NCM are disclosed above(FIGS. 3A-3J, 4A-4C). The NCM can be attached to both a non-dye-labeledoligonucleotide sequence which functions as the primer sequence as wellas to a dye-labeled oligonucleotide sequence which function as theprimer sequence. In some embodiments, the dye can be attached to anucleotide or nucleotide analog of the oligonucleotide sequence or tothe NCM as would be known to one of skill in the art.

The movement of DNA fragments under an electric field depends on thecharge to mass ratio. The DNA fragment can move differently by adding aNCM. Numerous NCM configurations and compositions attached to sequencingprimers were evaluated for their ability to change the amplifiedsequencing fragments mobility under an electric field. FIGS. 3A-3J,4A-4C illustrate exemplary M13 oligonucleotide sequence primers andgene-specific primers with various NCMs. The chromatograms illustratingthe results of sequencing reactions with the exemplaryNCM+oligonucleotide sequence primer structures with a variety oftemplates can be found in FIGS. 3-12 and 16 of U.S. Ser. No. 61/407,899,filed Oct. 28, 2011 and U.S. Ser. No. 61/408,553, filed Oct. 29, 2011).

In various embodiments, “analogs” in reference to nucleosides/tidesand/or polynucleotides comprise synthetic analogs having modifiednucleobase portions, modified pentose portions and/or modified phosphateportions, and in the case of polynucleotides, modified internucleotidelinkages, as described generally elsewhere (e.g., Scheit, NucleotideAnalogs (John Wiley, New York, (1980); Englisch, Angew. Chem. Int. ed.Engl. 30:613-29 (1991); Agrawal, Protocols for Polynucleotides andAnalogs, Humana Press (1994)). Generally, modified phosphate portionscomprise analogs of phosphate wherein the phosphorous atom is in the +5oxidation state and one or more of the oxygen atoms is replaced with anon-oxygen moiety e.g., sulfur. Exemplary phosphate analogs include butare not limited to phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, boronophosphates, includingassociated counterions, e.g., H⁺, NH₄ ⁺, Na⁺, if such counterions arepresent. Exemplary modified nucleobase portions include but are notlimited to 2,6-diaminopurine, hypoxanthine, pseudouridine, C-5-propyne,isocytosine, isoguanine, 2-thiopyrimidine, and other like analogs.Particularly preferred nucleobase analogs are iso-C and iso-G nucleobaseanalogs available from Sulfonics, Inc., Alachua, Fla. (e.g., Benner, etal., U.S. Pat. No. 5,432,272). Exemplary modified pentose portionsinclude but are not limited to 2′- or 3′-modifications where the 2′- or3′-position is hydrogen, hydroxy, alkoxy, e.g., methoxy, ethoxy,allyloxy, isopropoxy, butoxy, isobutoxy and phenoxy, azido, amino oralkylamino, fluoro, chloro, bromo and the like. Modified internucleotidelinkages include phosphate analogs, analogs having achiral and unchargedintersubunit linkages (e.g., Sterchak, E. P., et al., Organic Chem,52:4202 (1987)), and uncharged morpholino-based polymers having achiralintersubunit linkages (e.g., U.S. Pat. No. 5,034,506). A particularlypreferred class of polynucleotide analogs where a conventional sugar andinternucleotide linkage has been replaced with a 2-aminoethylglycineamide backbone polymer is peptide nucleic acid (PNA) (e.g., Nielsen etal., Science, 254:1497-1500 (1991); Egholm et al., J. Am. Chem. Soc.,114: 1895-1897 (1992)).

According to various embodiments, the chemically-enhanced primer cancomprise a universal primer, selected from US1, M13, T7, SP6, T3, orother sequencing primers as would be known to one of skill in the art.For example, an M13 universal forward primer, an M13 universal reverseprimer, or the like. In other embodiments, the chemically-enhancedprimer can comprise an oligonucleotide sequence or a specific genespecific oligonucleotide sequence, to the target nucleic acid sequencefor which the nucleic acid sequence was to be determined. A genespecific oligonucleotide sequence may be substantially hybridizable to aregion of a target nucleic acid on either the 5′ or 3′ side of a locusof interest. The oligonucleotide primer sequence can be the identicalprimer sequence as the sequence of the amplification primer used togenerate the PCR amplification product having the amplified nucleic acidtarget sequence and/or DNA target sequence.

In some embodiments, the chemically-enhanced primer may include an genespecific oligonucleotide sequence that is substantially hybridizable toa region of a target nucleic acid which encompasses a locus of interest.

While embodiments of a method for preparing nucleic acid for sequencingcan comprise using a phosphorothioated sequencing primer, and theteachings disclosed herein exemplify using a sequence primer having aNCM at or near the terminal 5′ end and a phosphorothioated terminal 3′end of an oligonucleotide sequence, other types of chemically-enhancedprimers can be utilized within the scope of the present teachings. Forexample, a nuclease resistant sequencing primer can comprise an alkylphosphonate monomer, RO—P(═O)(-Me)(—OR), such as dA-Me-phosphonamidite,and/or a triester monomer, RO—P(═O)(—OR′)(—OR), such asdA-Me-phosphoramidite (available from Glen Research, Sterling, Va.),and/or a locked nucleic acid monomer (available from Exiqon, Woburn,Mass.), and/or a boranophosphate monomer, RO—P(-BH₃)(═O) (—OR), asdescribed by Shaw, Barbara Ramsey, et al., in “Synthesis ofBoron-Containing ADP and GDP Analogues: Nucleoside5′-(P-Boranodisphosphates)”, Perspectives in Nucleoside and Nucleic AcidChemistry, pg. 125-130, (2000), or the like.

According to various embodiments, the amplification reaction productscan comprise a target amplicon. In some embodiments, the target ampliconcan comprise a result of PCR amplification from amplification primers.In some embodiments, the target amplicon can comprise double strandedDNA. In some embodiments, the target amplicon can comprise singlestranded DNA.

According to various embodiments, the amplification primers can comprisetailed primers. The tailed primers can be used, for example, to generatea target specific amplicon that incorporates nucleic acid sequencecapable of annealing to a universal primer or a gene specific primer.

According to various embodiments, a method for preparing nucleic acidfor sequencing can comprise inactivating a nuclease after excess primeris degraded by the nuclease. In some embodiments, the nuclease can beinactivated by heating. For example, the nuclease can beheat-inactivated by heating to a temperature of from about 80° C. toabout 90° C. for about 15 minutes. In various embodiments, theinactivation of the nuclease can occur within the vesicle and in thesame reaction step as the sequencing reaction as shown in FIG. 1B in theCycle Seq. (cycle sequencing) step.

According to various embodiments of the present teachings, templates tobe sequenced can be synthesized by PCR in individual aqueouscompartments (also called “reactors”) of an emulsion. In someembodiments, the compartments can each contain a particulate supportsuch as a bead having a suitable first amplification primer attachedthereto, a first copy of the template, a second amplification primer,and components needed for a PCR reaction (for example nucleotides,polymerase, cofactors, and the like). Methods for preparing emulsionsare described, for example, in U.S. Pat. No. 6,489,103 B1, U.S. Pat. No.5,830,663, and in U.S. Patent Application Publication No. US2004/0253731. Methods for performing PCR within individual compartmentsof an emulsion to produce clonal populations of templates attached tomicroparticles are described, for example, in Dressman, D., et al, Proc.Natl. Acad. Sci., 100(15):8817-8822, 2003, and in PCT publicationWO2005010145. All of the patents, applications, publications, andarticles described herein are incorporated in their entireties byreference.

According to various embodiments, a method for sequencing nucleic acidcan comprise amplifying nucleic acid in a first reaction mixturecomprising nuclease sensitive amplification primers to form amplifiednucleic acid, contacting the first reaction mixture with a secondreaction mixture comprising a nuclease and a chemically-enhanced primer,under conditions in which the nuclease sensitive amplification primersare degraded by the nuclease, and inactivating the nuclease and causingthe amplified nucleic acid to react in a sequencing reaction underconditions in which the chemically-enhanced primer primes the sequencingreaction. According to various embodiments, results can be obtainedbased on the sequencing reaction and a nucleotide base sequence of theamplified nucleic acid can be determined based on the results. Accordingto various embodiments, the nucleotide base sequence can be determinedby a mobility-dependent separation of the sequencing reaction products.According to various embodiments, the amplifying can be by polymerasechain reaction amplification.

According to various embodiments, the second reaction mixture canfurther comprise dNTPs, ddNTPs, a dye-label, and a thermo-stable DNApolymerase. In some embodiments, each of the ddNTPs can be labeled witha different fluorescent dye (ddNTP-dye). For example, the ddNTPs cancomprise BigDye ddNTPs, available from Applied Biosystems, Foster City,Calif. In some embodiments, the chemically-enhanced primer can belabeled with a fluorescent dye. The label can be attached to theoligonucleotide sequence and/or the NCM region of thechemically-enhanced primer. The thermo-stable DNA polymerase cancomprise, for example, a DNA polymerase known to one of skill in theart. In some embodiments, the sequencing reaction can comprise a thermalcycle sequencing reaction.

In other embodiments, the chemically-enhanced primers can be utilized ina sequencing reaction performed without an initial amplification step.Such sequencing methods may sequence either single or doubled strandednucleic acids. For example, plasmid sequencing may be more efficientlyperformed using one or both of a primer pair having the structure of achemically-enhanced primer as disclosed herein. In some embodiments, achemically-enhanced primer may have inter-nucleotide linkages that areall phosphodiester linkages. In other embodiments of plasmid sequencingmethods, a chemically-enhanced primer may have at least onephosphothiorate inter-nucleotide linkage. In some embodiments, thechemically-enhanced primer has one phosphothioate linkage.

In another method, one or more chemically-enhanced primers may be usedfor ligation extension reactions. In some embodiments, thechemically-enhanced primer for use in a ligation extension reaction islabeled fluorescently. In some embodiments, the ligation extensionchemically-enhanced primer is labeled fluorescently at a 3′ terminus.

A variety of nucleic acid polymerases may be used in the methodsdescribed herein. For example, the nucleic acid polymerizing enzyme canbe a thermostable polymerase or a thermally degradable polymerase.Suitable thermostable polymerases include, but are not limited to,polymerases isolated from Thermus aquaticus, Thermus thermophilus,Pyrococcus woesei, Pyrococcus furiosus, Thermococcus litoralis, andThermotoga maritima. Suitable thermodegradable polymerases include, butare not limited to, E. coli DNA polymerase I, the Klenow fragment of E.coli DNA polymerase I, T4 DNA polymerase, T5 DNA polymerase, T7 DNApolymerase, and others. Examples of other polymerizing enzymes that canbe used in the methods described herein include T7, T3, SP6 RNApolymerases and AMV, M-MLV and HIV reverse transcriptases.

Non-limiting examples of commercially available polymerases that can beused in the methods described herein include, but are not limited to,TaqFS®, AmpliTaq® CS (Applied Biosystems), AmpliTaq FS (AppliedBiosystems), AmpliTaq Gold® (Applied Biosystems), Kentaq1 (AB Peptide,St. Louis, Mo.), Taquenase (ScienTech Corp., St. Louis, Mo.),ThermoSequenase (Amersham), Bst polymerase, Vent_(R)(exo⁻) DNApolymerase, Reader™ Taq DNA polymerase, VENT™ DNA polymerase (NewEngland Biolabs), DEEPVENT™ DNA polymerase (New England Biolabs),PFUTurbo™ DNA polymerase (Stratagene), Tth DNA polymerase, KlenTaq-1polymerase, SEQUENASE™ 1.0 DNA polymerase (Amersham Biosciences), andSEQUENASE 2.0 DNA polymerase (United States Biochemicals).

According to various embodiments of a method for sequencing nucleicacid, the nuclease can comprise exonuclease I. The exonuclease I can besensitive to heat inactivation and can be essentially 100 percentdeactivated by heating, for example, heating at about 80° C. for about15 minutes. Other heat inactivated nucleases may be used in the subjectmethods and compositions including but not limited to Exo III, Pfu orDNA pol I.

According to various embodiments, the chemically-enhanced primercomprises at least one phosphorothioate linkage. In some embodiments,the chemically-enhanced primer comprises at least one terminal 3′ endphosphorothioate linkage. In some embodiments, as described above, thechemically-enhanced primer comprises a NCM and an oligonucleotidesequence 5′ of the phosphorothioate linkage.

The sequencing reaction products can be analyzed on a sieving ornon-sieving medium. In some embodiments of these teachings, for example,the PCR products can be analyzed by electrophoresis; e.g., capillaryelectrophoresis, as described in H. Wenz et al. (1998), GENOME RES.8:69-80 (see also E. Buel et al. (1998), J. FORENSIC SCI. 43:(1), pp.164-170)), or slab gel electrophoresis, as described in M. Christensenet al. (1999), SCAND. J. CLIN. LAB. INVEST. 59(3): 167-177, ordenaturing polyacrylamide gel electrophoresis (see, e.g., J. Sambrook etal. (1989), in MOLECULAR CLONING: A LABORATORY MANUAL, SECOND EDITION,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp.13.45-13.57). The separation of DNA fragments in electrophoresis isbased primarily on differential fragment size. Sequencing reactionproducts can also be analyzed by chromatography; e.g., by size exclusionchromatography (SEC). Likewise, fragment analysis can be carried in asimilar manner as would be known to the skilled artisan.

According to various embodiments a system for sequencing DNA cancomprise amplifying DNA in a first reaction mixture comprising nucleasesensitive amplification primers to form amplified DNA; contacting saidfirst reaction mixture of the amplifying step with a second reactionmixture comprising a nuclease and a chemically-enhanced primer, underconditions in which the nuclease sensitive amplification primers aredegraded by the nuclease; inactivating the nuclease and causing theamplified DNA to react in a sequencing reaction under conditions inwhich the chemically-enhanced primer primes said sequencing reaction;and identifying a nucleotide base sequence of the amplified DNA bymobility-dependent separation of sequencing reaction products. Invarious embodiments, the system for sequencing DNA, themobility-dependent separation is selected from separation by charge andseparation by size, wherein the separation by size plus charge isselected from gel electrophoresis and capillary electrophoresis andseparation by size is by a liquid gradient, and a denaturing gradientmedium.

The present teachings are also directed to kits that utilize thechemically-enhanced primer composition and methods described above. Insome embodiments, a basic kit can comprise a container having one ormore chemically-enhanced primers. A kit can also optionally compriseinstructions for use. A kit can also comprise other optional kitcomponents, such as, for example, one or more of a nuclease, asufficient quantity of enzyme for sequencing or fragment analysis,buffer to facilitate the sequencing reaction or fragment analysisreaction, dNTPs, modified dNTPs, dNTP analogs and 7-Deaza-dGTP forstrand extension during sequencing reaction or fragment analysisreaction, ddNTPs, a dye-label, loading solution for preparation of thesequenced or fragment analyzed material for electrophoresis, genomic DNAas a template control, a size marker to insure that materials migrate asanticipated in the separation medium, and a protocol and manual toeducate the user and limit error in use. The amounts of the variousreagents in the kits also can be varied depending upon a number offactors, such as the optimum sensitivity of the process. It is withinthe scope of these teachings to provide test kits for use in manualapplications or test kits for use with automated detectors or analyzers.Kits may have more than one chemically enhanced primer, and the numberof NCM moieties may be different in each of the chemically enhancedprimers. Kits for plasmid sequencing may have any of the componentslisted above, but do not include a nuclease.

Examples of the compositions and methods of the present teachings areshown below. These examples are not limiting of the present teachings,and those of ordinary skill in the art will recognize that thecomponents used in the reactions may be readily substituted withequivalent reagents known in the art.

The following Examples illustrate the ability of the chemically-enhancedprimer to provide higher resolution of a sequencing reaction and in lesstime with POP7 polymer and variants thereof, the stability of thechemically-enhanced primer to exonuclease I, the incorporation of thechemically-enhanced primer as a substrate for DNA polymerase, thecompatibility of exonuclease I with the sequencing reagents, and thesusceptibility of non-phosphorothioate primer to exonuclease Idigestion. The Examples further illustrate the use of tailedamplification primers along with universal phosphorothioate primers forsequencing.

Example 1

C6 spacer+Oligo Seq. Synthesis, No Phosphorothioate Group:

An 18 base oligonucleotide labeled with one or more C6 spacers at the 5′position was made on an ABI model 394 DNA synthesizer using standardphosphoramidite chemistry. The C6 spacer phosphoramidite was obtainedfrom Chem Genes Corp. (P/N CLP-1120, Wilmington, Mass.). The labeled18mer was made with the trityl group intact from a one micromole column.On completion of the synthesis the oligonucleotide was cleaved off thesupport with NH₄OH and purified by HPLC using an ABI RP-300 (C-8) column(4.6×220 mm) using a flow rate of 1.5 ml/min. and a solvent gradient of0.1M triethylammonium acetate-water pH 7.0 and acetonitrile, the tritylgroup was removed and the product was isolated by ethanol precipitation.

C3 Spacer+Oligo Seq. Synthesis, No Phosphorothioate Group:

An 18 base oligonucleotide labeled with one or more C3 spacers (P/N10-1913-90, Glen Research), at the 5′ position was made on an ABI model394 DNA synthesizer using standard phosphoramidite chemistry. Thelabeled 18mer was made with the trityl group intact from a one micromolecolumn. On completion of the synthesis the oligonucleotide was cleavedoff the support with NH₄OH and purified by HPLC using an ABI RP-300(C-8) column (4.6×220 mm) using a flow rate of 1.5 ml/min. and a solventgradient of 0.1M triethylammonium acetate-water pH 7.0 and acetonitrile,the trityl group was removed and the product was isolated by ethanolprecipitation.

Protocol for Oligo Labeled with a 5′ Phosphate:

An 18 base oligonucleotide labeled with a phosphate group at the 5′position. This was made on an ABI model 394 DNA synthesizer usingstandard phosphoramidite chemistry. The phosphate group was generatedusing a phosphoramidite obtained from Glen Research (P/N 10-1922-90) Thelabeled 18mer was made from a one micromole column and on completion ofthe synthesis the oligonucleotide was cleaved off the support with NH₄OHand purified by HPLC using an ABI RP-300 (C-18) column (4.6×220 mm)using a flow rate of 1.5 ml/min. and a solvent gradient of 0.1Mtriethylammonium acetate-water pH 7.0 and acetonitrile. The product wasthen isolated by ethanol precipitation.

Protocol for Oligo Labeled with Dual Branching (Doubler) Linker Labeledwith One or More C3 Spacers:

An 18 base oligonucleotide labeled with a dual branching linkagefollowed by one or more C3 spacers at the 5′ position was made on an ABImodel 394 DNA synthesizer using standard phosphoramidite chemistry. Thedual (doubler) branching (P/N 10-1920-90) and C3 spacer (P/N 10-1913-90)phosphoramidites were obtained from Glen Research. The labeled 18mer wasmade with the trityl group intact using a one micromole synthesiscolumn. On completion of the synthesis the oligonucleotide was cleavedoff the support with NH₄OH and purified by HPLC using an ABI RP-300(C-18) column (4.6×220 mm) using a flow rate of 1.5 ml/min. and asolvent gradient of 0.1M triethylammonium acetate-water pH 7.0 andacetonitrile, the trityl group was removed and the product was isolatedby ethanol precipitation.

Protocol for Oligo Labeled with Trebler Branching (Trebler) LinkerLabeled with One or More C3 Spacers:

An 18 base oligonucleotide labeled with a trebler branching linkagefollowed by one or more C-3 spacers at the 5′ position was made on anABI model 394 DNA synthesizer using standard phosphoramidite chemistry.The trebler phosphoramidite (P/N 10-1922-90) and C-3 spacer (P/N10-1913-90) phosphoramidites were obtained from Glen Research. Thelabeled 18mer was made with the trityl group intact using a onemicromole synthesis column. On completion of the synthesis theoligonucleotide was cleaved off the support with NH₄OH and purified byHPLC using an ABI RP-300 (C-18) column (4.6×220 mm) using a flow rate of1.5 ml/min. and a solvent gradient of 0.1M triethylammoniumacetate-water pH 7.0 and acetonitrile, the trityl group was removed andthe product was isolated by ethanol precipitation.

Protocol for Oligo Labeled with Trebler Branching Linker End Labeledwith Phosphates (3 Total Phosphates):

An 18 base oligonucleotide labeled with a trebler branching linkage atthe 5′ position followed by phosphorylation was made on an ABI model 394DNA synthesizer using standard phosphoramidite chemistry. The treblerbranching (P/N 10-1922-90) and phosphorylating (P/N 10-1900-90)phosphoramidites were obtained from Glen Research. The labeled 18mer wasmade using a one micromole synthesis column. On completion of thesynthesis the oligonucleotide was cleaved off the support with NH₄OH andpurified by HPLC using an ABI RP-300 (C-18) column (4.6×220 mm) using aflow rate of 1.5 ml/min. and a solvent gradient of 0.1M triethylammoniumacetate-water pH 7.0 and acetonitrile. The product was isolated byethanol precipitation.

Protocol for Oligo Labeled with Two Generations of Trebler BranchingLinker End Labeled with Phosphates (9 Total Phosphates):

An 18 base oligonucleotide labeled with 2 additions of trebler branchinglinkages at the 5′ position followed by phosphorylation was made on anABI model 394 DNA synthesizer using standard phosphoramidite chemistry.The trebler branching (P/N 10-1922-90) and phosphorylating (P/N10-1900-90) phosphoramidites were obtained from Glen Research. Thelabeled 18mer was made using a one micromole synthesis column. Oncompletion of the synthesis the oligonucleotide was cleaved off thesupport with NH₄OH and purified by HPLC using an ABI RP-300 (C-18)column (4.6×220 mm) using a flow rate of 1.5 ml/min. and a solventgradient of 0.1M triethylammonium acetate-water pH 7.0 and acetonitrile.The product was isolated by ethanol precipitation.

Protocol for Oligo Labeled with One or More C-3 Spacer Containing a 3′Phosphorothioate Linkage:

An 18 base oligonucleotide labeled with one or more C-3 spacers at the5′ position was made on an ABI model 394 DNA synthesizer using standardphosphoramidite chemistry. The 3′ phosphorothioate linkage was madeusing standard methods with sulfurizing reagent (TETD P/N 401267(Applied Biosystems, Foster City, Calif.). The C3 spacer phosphoramiditewas obtained from Glen Research (P/N 10-1913-90). The labeled 18mer wasmade with the trityl group intact from a one micromole synthesis column.On completion of the synthesis the oligonucleotide was cleaved off thesupport with NH₄OH and purified by HPLC using an ABI RP-300 (C-18)column (4.6×220 mm) using a flow rate of 1.5 ml/min. and a solventgradient of 0.1M triethylammonium acetate-water pH 7.0 and acetonitrile,the trityl group was removed and the product was isolated by ethanolprecipitation. Note: To synthesize more than one phosphorothioatelinkage or to place this linkage anywhere in the 18-mer oligonucleotidechain, oxidize using the sulfurizing reagent at these position(s).

Example 2 PCR and Sequencing Reactions:

PCR and Sequencing Reactions Using a Chemically-Enhanced Primer with aterminal 3′ Phosphorothioate (PS) Linkage

PCR Amplification

PCR reactions were carried out in the following 10 μL solution: tosequence amplicon RSA000013703, 1 μL 4 ng/μL gDNA, M13-tagged primers(0.8 uM each): 1.5 μL of TGTAAAACGACGGCCAGTTTGATGGGCTCAGCAACAGGT (SEQ IDNO:1, gnl|Probe|1292199b) and CAGGAAACAGCTATGACCCCACTGCTTGCGTTTCTTCCTG(SEQ ID NO:2, gnl|Probe|1292199c), 5 μL of BigDye Direct PCR Master Mix(P/N 4458699, Applied Biosystems) and 2.5 μL water. PCR was carried outon a Veriti™ 96-well thermal cycler (P/N 4375786, Applied Biosystems).with the following thermal cycling conditions: 95° C. for 10 minutes,then 35 cycles of 96° C. for 3 seconds, 62° C. for 15 seconds, and 68°C. for 30 sec. followed by 72° C. for 2 min. and 4° C. hold. To confirmamplification a 10 μl sample was analyzed on an agarose gel. A bandconsistent with the expected 626 bp amplicon was observed.

Amplicon RSA000013703 SEQ ID NO:3, template ZC is shown below withprimer binding sites for PCR forward primer, and PCR reverse primer(reverse complement) underlined.

CAGGAAACAGCTATGACCCCACTGCTTGCGTTTCTTCCTGTTTTAATCCCACTTTCAATGAAGTGTGTATTTGAAATAAATGGCTCATGAGTTAATCACATCTTTATATATCCTAAGATGTATTACAAAGGCTTCCATAACACTTGTCTATAGTAAGCCACTCATTTCTATAATTTTTCTTTCAATAAACTCAATCTTTGTAATACAGAAATTAACCTTCTGGGTTGTTTTTGTTCAAGATCTTCAGTTTGATTTGCCCCTTGGTTGATCTGTTTTTCCCATCGCTGAACTGGTTCCCATAATCACACACCTTTGCTTTTCATTTCCACAGATCAAGGAATCAACATTTACCGAAAGCCACCCATCTACAAACAGCATGGTAAAACCCGCTTTCCTCCGCGTAGCTTTTAAATAGCAAAGTCAGCTGAACTTCTCCTTGCTGTCCTCTGAAAGGCTTTTCCTGCTGCTGCTTTTGAGAGTAAAACTGGGGCATCCAGCATATTATGCCTTTCTGGTCTACTAAGATGTAAATATTGTAAAATTGATTCTCCTGGATGGAGAGACTTAGCTTGATTAGAAAGCTTCTAACCTGTTGCTGAGCCCATCAAACTGGCCGTCGTTTTACA.

Sequencing Workflow with a Chemically Enhanced Primer Having aPhosphorothioate (PS) Linkage

Sequencing reaction prepared with the BigDye Direct Cycle Sequencing Kit(24 reactions, P/N 4458689, Applied Biosystems): 10 μL of the PCRamplification reaction was mixed with 2 μl BigDye® Direct SequencingMaster Mix (P/N 4458701, Applied Biosystems), and 1 μl BigDye Direct M13Forward Primer (P/N 4458692 Applied Biosystems) or BigDye Direct M13Reverse Primer 4458695, Applied Biosystems). The BigDye Direct Primershave the terminal 5′ NCM and a terminal 3′ PS. The cycle sequencingreaction was carried out on a Verti 96-well thermal cycler at 37° C. 15min. at which point excess PCR amplification primers were digested bythe Exol nuclease (contained in the BigDye Direct Sequencing MasterMix), then 80° C. 2 min, 96° C. 1 min. followed by 25 cycles, of 96° C.10 seconds, 50° C. 5 seconds, and 60° C. 1 minute 15 seconds and 4° C.hold. The Sequencing primer contained a terminal 5′ Negatively ChargedMoiety (NCM) and a terminal 3′ phosphorothioate group indicated by anasterisk: M13 forward primer (1 μM) (NCM-TGTAAAACGACGGCCAG*T) (SEQ IDNO:4) or M13 reverse primer (1 μM) (NCM-CAGGAAACAGCTATGAC*C) (SEQ IDNO:5). FIG. 3H provides the structure of the NCM. FIG. 11 is aelectropherogram (see U.S. Ser. No. 61/407,899, filed Oct. 28, 2011 andU.S. Ser. No. 61/408,553, filed Oct. 29, 2011), of a chemically-enhancedprimer (FIG. 3H) with a terminal 3′ PS linkage and ZC as the template.The sequence of ZC shows high resolution at base 1 from the primer.

Example 3

PCR reactions were carried out in the following 10 μL solution: tosequence amplicon RSA000317141 (Template Seq01, 545 bp), 1 μL 4 ng/μLgDNA, M13-tagged primers (0.8 uM each): 1.5 μL ofTGTAAAACGACGGCCAGTGCTGCCTCTGATGGCGGAC (SEQ ID NO:6, forward,gnl|Probe|1204459b) and CAGGAAACAGCTATGACCGCCACACTCTGGAGCTGGACA (SEQ IDNO:7, reverse, gnl|Probe|1204459c), 5 μl of BigDye Direct PCR Master Mix(P/N 4458699, Applied Biosystems) and 2.5 μl water. PCR was carried outon a Veriti™ 96-well thermal cycler (P/N 4375786, Applied Biosystems).with the following thermal cycling conditions: 95° C. for 10 minutes,then 35 cycles of 96° C. for 3 seconds, 62° C. for 15 seconds, and 68°C. for 30 sec. followed by 72° C. for 2 min. and 4° C. hold. To confirmamplification a 10 μl sample was analyzed on an agarose gel. A bandconsistent with the expected 545 bp amplicon was observed.

Amplicon RSA000317141 SEQ ID NO:8, template Seq01 (FIG. 3I) is shownbelow with primer binding sites for PCR forward primer, and PCR reverseprimer (reverse complement) underlined.

TGTAAAACGACGGCCAGTGCTGCCTCTGATGGCGGACGGGGGTGTGGTCCTGGGACTCGTGGTCAGGGCTGGTCTGTGTGGAATGCTGATCCTTCTCTTCCCCAATCTACCTGTGTCAGTTCCCTCCTTTTCTATTTTCTCTTCCCTGCAGATGTCAAGCCCTCCAACATCCTAGTCAACTCCCGTGGGGAGATCAAGCTCTGTGACTTTGGGGTCAGCGGGCAGCTCATCGACTCCATGGCCAACTCCTTCGTGGGCACAAGGTCCTACATGTCGGTATGAACAGAAGTTTCCATTGCTTGAGCTTCTTGTACGGTCAGGGAGAGGAGCCCAGTGGGTGCCTTTCCTGTGGAGCCAGAGTCTTGTGCTGGGTAGGGGACAAGAAGTGAGGGAGGAGGCACAGTGCTCTGCCCTGAGGAGATGAAGTTGAATGGGAAGATGGTCTTGGTCTTTCTTAGGCCTTGGAGCATAACTGGGATATTGGGGCCTTGACTCACTGAAAGGACTGTCCAGCTCCAGAGTGTGGCGGTCATAGCTGTTTCCTG.

Sequencing Workflow with a Chemically-Enhanced Primer Having aPhosphorothioate (PS) Linkage.

Sequencing reaction prepared with the BigDye Direct Cycle Sequencing Kit(24 reactions, P/N 4458689, Applied Biosystems): 10 μL of the PCRamplification reaction was mixed with 2 μL BigDye® Direct SequencingMaster Mix (P/N 4458701, Applied Biosystems), and 1 μl BigDye Direct M13Forward Primer (P/N 4458692 Applied Biosystems) or BigDye Direct M13Reverse Primer 4458695, Applied Biosystems). The BigDye Direct Primershave the terminal 5′ NCM and a terminal 3′ PS. The cycle sequencingreaction was carried out on a Verti 96-well thermal cycler at 37° C. 15min. at which point excess PCR amplification primers were digested bythe Exol nuclease (contained in the BigDye Direct Sequencing MasterMix), then 80° C. 2 min, 96° C. 1 min. followed by 25 cycles, of 96° C.10 seconds, 50° C. 5 seconds, and 60° C. 1 minute 15 seconds and 4° C.hold. The Sequencing primer contained a terminal 5′ Negatively ChargedMoiety (NCM) and a terminal 3′ phosphorothioate group indicated by anasterisk: M13 forward primer (1 μM) (NCM-TGTAAAACGACGGCCAG*T) (SEQ IDNO:4) or M13 reverse primer (1 μM) (NCM-CAGGAAACAGCTATGAC*C) (SEQ IDNO:5). FIG. 16 (see U.S. Ser. No. 61/408,553, filed Oct. 29, 2011),provides a electropherogram of a chemically-enhanced primer with aterminal 3′ PS linkage and RSA000317141 as the template (FIG. 3J). Thesequence of RSA000317141 shows high resolution at base 1 from theprimer.

Example 4

PCR and Sequencing Reactions with or without PS Linkage:

Standard PCR Amplification: PCR reactions were carried out in thefollowing 10 μL solution: for example to sequence amplicon RSA0003176671μL 10 ng/μL gDNA, primers (0.8 uM each), 1.5 μL ofTGTAAAACGACGGCCAGTGGCTCCTGGCACAAAGCTGG (gnl|Probe|1172813b, forward, SEQID NO:9) and CAGGAAACAGCTATGACCTGCATCTCATTCTCCAGGCTTC(gnl|Probe|1172813c, reverse, SEQ ID NO:10), 5 μL of AmpliTaq Gold® PCRMaster Mix (P/N 4318739, Applied Biosystems), 1.6 μL 50% glycerol and0.9 μl water. PCR was carried using a Gold-plated 96-Well GeneAmp® PCRSystem 9700 (P/N 4314878, Applied Biosystems). Thermal cyclingconditions: 96° C. 5 minutes, then 40 cycles of 94° C. 30 seconds, 60°C. 45 seconds, and 72° C. 45 sec. followed by 72° C. 2 min. and 4° C.hold. A 5 μL aliquot was analyzed on an agarose gel. A band consistentwith the expected 630 bp amplicon was observed.

PCR Clean-Up:

The 10 μl of PCR amplification reaction was mixed with 2 μL ofExoSAP-IT® nuclease (P/N 78250, Affymetrix, Santa Clara, Calif.) andincubated on a Gold-plated 96-Well GeneAmp® PCR System 9700 (P/N4314878, Applied Biosystems) at 37° C. 30 minutes followed by 80° C. for15 minutes (inactivated the nuclease.

Sequencing Workflow with a Chemically-Enhanced Primer withoutPhosphorothioate Linkage

A sequencing reaction was prepared with the BigDye® Terminator v3.1Cycle Sequencing Kit (24 reactions, P/N 4337454, Applied Biosystems): 2μL of the PCR amplification reaction treated with ExoSAP-IT was mixedwith 4 μL BigDye® Terminator v3.1 Cycle Sequencing Kit Master Mix (P/N4337454, Applied Biosystems), 1 μL Sequencing primerschemically-enhanced with terminal 5′ NCM and with or without a terminal3′ phosphorothioate linkage, (NCM-M13 forward and NCM-M13 reverseprimer) and 3 μL water. The cycle sequencing reaction was carried out at96° C. 1 min. followed by 25 cycles, 96° C. 10 sec. 50° C. 5 sec, 60° C.1 min. 15 sec followed by 4° C. hold. For example, the sequencing primeris M13 forward primer (1 μM) containing a terminal 3′ phosphorothioate(PS) group indicated by an asterisk (NGM-TGTAAAACGACGGCCAG*T) (SEQ IDNO:4), M13 reverse primer (1 μM) (NGM-CAGGAAACAGCTATGAC*C) (SEQ ID NO:5)or without PS, M13 forward primer (1 μM) (NGM-TGTAAAACGACGGCCAGT) (SEQID NO:11), M13 reverse primer (1 μM) (NGM-CAGGAAACAGCTATGACC) (SEQ IDNO:12). FIGS. 4 and 8 provide electropherograms (see U.S. Ser. No.61/407,899, filed Oct. 28, 2011 and U.S. Ser. No. 61/408,553, filed Oct.29, 2011), of chemically-enhanced sequencing primers (FIGS. 3A and 3F,respectively), with terminal 5′ negatively charged moieties, without PSand RSA000317667 as the template. The sequences for RSA000317667 showhigh resolution at base 1 from the primer.

Example 5

Capillary Electrophoresis Sample Preparation and Detection

The amplified samples are analyzed by methods that resolve nucleobasesequences as would be known to one of skill in the art. For example,capillary electrophoresis can be used following the instrumentmanufactures directions. BigDye XTerminator Purification Kit (AppliedBiosystems, P/N 4376486) can be used in cycle sequencing clean up toprevent the co-injection of un-incorporated dye-labeled terminators,dNTPs and salts with dye-labeled extension products into a capillaryelectrophoresis DNA analyzer. Briefly, 13° L sequencing reaction mixturewas combined with 45 μL SAM Solution and 10 μL XTerminator Solution.After vortexing the sample plate at 1800 rpm for 20 minutes, spin theplate at 1000×g for 2 minutes. To each well was added 30 μL of 70%ethanol and the plate was centrifuged at 1650×g for 15 minutes. Thesolution was removed by inverting the plate onto a paper towel andcentrifuging at 180×g for 1 minute. The precipitated sequencing reactionwas then dissolved in 10 μl of 50 μM EDTA and loaded onto an AB 3500xLGenetic Analyzer equipped with a 50 cm capillary array (AppliedBiosystems, Foster City, Calif.).

Example 6

Capillary Electrophoresis Methods and Analysis

Capillary electrophoresis (CE) was performed on the current AppliedBiosystems instruments, for example the Applied Biosystems 3500×1Genetic Analyzer, using the dye set Z as described the instrument's UserGuide. There are ShortReadSeq_BDX_POP7, RapidSeq_BDX_POP7,FastSeq_BDX_POP7, StdSeq_BDX_POP7 run modules. For example,BDxFastSeq50_POP7xl_(—)1 parameters were: oven temperature: 60 C, sampleinjection for 5 sec at 1.6 kV and electrophoresis at 13.4 kV for 2520sec in Performance Optimized Polymer (POP-7™ polymer) with a runtemperature of 60° C. Variations in instrument parameters, e.g.injection conditions, were different on other CE instruments such as the3500 or 3730xl Genetic Analyzers. The data were collected using versionsthe Applied Biosystems Data Collection Software specific to thedifferent instruments, such as 3500 Data Collection Software v1.0. Thesequence traces were analyzed by Applied Biosystems KB™ BasecallerSoftware v1.4.1 with KB_(—)3500_POP7-BDTv3direct.bcc andKB_(—)3500_POP7-BDTv3direct.mob to determine the correct base calls.

Example 7

Sequencing of Plasmid DNA or PCR Amplified Exo/SAP treated DNA

200 ng of pGem plasmid DNA or long of Exo/SAP treated PCR product mixedwith BigDye Terminator v3.1 Ready Reaction reagent and sequencing primerhaving an NCM composition without PS was reacted in a cycle sequencingreaction: 95 C/1 min and 25 cycles of (96C/1 min, 50C/5 sec, 60 C./1 min15 sec). The reaction was cleaned up using BigDye XTerminator Kit(Applied Biosystems) and electrophrosesed on the 3500 with POP7 polymer.The electropherogram of the sequenced pGEM plasmid sequenced using thesequencing primer having NCM on the 5′ indicated clear sequence withinfive base pairs of the primer (data not shown). The electropherogram ofthe sequenced pGEM plasmid sequenced using the sequencing primer havingNCM on the 5′ and nuclease resistance linkage on the 3′ end indicatedclear sequence five base pairs after the primer (data not shown). Theelectropherogram of the Exo/SAP treated PCR product sequenced using thesequencing primer with NCM on the 5′ and nuclease resistance linkage onthe 3′ end indicated clean sequence reads six bases after the primer(data not shown).

Those skilled in the art understand that the detection techniquesemployed are generally not limiting. Rather, a wide variety of detectionmeans are within the scope of the disclosed methods and kits, providedthat they allow the presence or absence of an amplicon to be determined.

While the principles of this invention have been described in connectionwith specific embodiments, it should be understood clearly that thesedescriptions are made only by way of example and are not intended tolimit the scope of the invention. What has been disclosed herein hasbeen provided for the purposes of illustration and description. It isnot intended to be exhaustive or to limit what is disclosed to theprecise forms described. Many modifications and variations will beapparent to the practitioner skilled in the art. What is disclosed waschosen and described in order to best explain the principles andpractical application of the disclosed embodiments of the art described,thereby enabling others skilled in the art to understand the variousembodiments and various modifications that are suited to the particularuse contemplated. It is intended that the scope of what is disclosed bedefined by the following claims and their equivalence.

1. A chemically-enhanced primer having a structure of Formula I:

wherein B is a nucleobase; K is S or O; each instance of n is independently an integer of 1 to 9; m is 0 or 1; x is an integer of 1 to about 30; z is an integer of 3 to about 100; W is OH, F, OMe, or H; and Nt is a moiety having a formula:


2. The chemically-enhanced primer of claim 1, wherein m is
 0. 3. The chemically-enhanced primer of claim 1, wherein K is S.
 4. (canceled)
 5. The chemically-enhanced primer of claim 1, wherein the chemically-enhanced primer is fluorescently labeled.
 6. (canceled)
 7. The chemically-enhanced primer of claim 1, wherein W is H or OH.
 8. The chemically-enhanced primer of claim 1, wherein n is 3 or
 6. 9. (canceled)
 10. The chemically-enhanced primer of claim 1, wherein x is 5, 8, 9, 10, or
 15. 11. The chemically-enhanced primer of claim 1, wherein z is an integer of 5 to
 30. 12. The chemically-enhanced primer of claim 1, having a structure of one of the following formulae:

wherein FL is a dye label and B^(f) is a dye labeled nucleobase.
 13. The chemically-enhanced primer of claim 1, having an OLIGO portion of the following structure:

wherein OLIGO comprises a universal primer.
 14. The chemically-enhanced primer of claim 13, wherein the universal primer is selected from M13, US1, T7, SP6, and T3.
 15. The chemically-enhanced primer of claim 1, having an OLIGO portion wherein OLIGO comprises a gene specific oligonucleotide sequence. 16.-32. (canceled)
 33. A composition for sequencing nucleic acid comprising: a chemically-enhanced primer of Formula I:

wherein B is a nucleobase; K is S or O; each n is independently an integer of 1 to 9; m is 0 or 1; x is an integer of 1 to about 30; z is an integer of 3 to about 100; W is OH, F, OMe, or H; and Nt is a moiety having a formula:


34. The composition of claim 33, wherein the oligonucleotide portion of the chemically-enhanced primer comprises a universal primer.
 35. The composition of claim 34 wherein the universal primer is selected from M13, US1, T7, SP6, and T3.
 36. The composition of claim 33, wherein the chemically-enhanced primer comprises one nuclease-resistant linkage.
 37. The composition of claim 33, further comprising a polymerase, a nuclease, deoxynucleotide triphosphates, dideoxynucleotide triphosphates and a dye-label.
 38. The composition of claim 37, wherein the dideoxynucleotide triphosphates comprise dye-labeled dideoxynucleotide triphosphates.
 39. (canceled)
 40. The composition of claim 37, wherein the nuclease is selected from exonuclease I, Exo III, Pfu and DNA pol I.
 41. (canceled)
 42. The composition of claim 37, further comprising a PCR amplification reaction product that comprises non-nuclease-resistant amplification primer(s).
 43. The composition of claim 42, wherein the PCR amplification reaction product further comprises an amplified DNA target sequence.
 44. The composition of claim 37, wherein the polymerase is Taq polymerase.
 45. The composition of claim 35, wherein the universal primer is M13. 46.-73. (canceled)
 74. A kit comprising a chemically enhanced primer wherein the chemically enhanced primer has a structure of Formula I:

wherein B is a nucleobase; K is S or O; each instance of n is independently an integer of 1 to 9; m is 0 or 1; x is an integer of 1 to about 30; z is an integer of 3 to about 100; W is OH, F, OMe, or H; and Nt is a moiety having a formula:


75. The kit of claim 74, wherein the chemically-enhanced primer has a structure of the following formula: (Cn)_(x)OLIGO, wherein (Cn)_(x) has a structure of the following formula:

wherein each instance of n is independently an integer of 1 to 9; and x is an integer of 1 to about 30; OLIGO has a structure of the following formula:

wherein B is a nucleobase; K is S or O; m is 0 or 1; z is an integer of 3 to about 100; W is OH, F, OMe, or H; and Nt is a moiety having a formula:


76. The kit of claim 75, wherein the chemically enhanced primer is selected from the group consisting of Cn)_(x)-US1, (Cn)_(x)-M13-forward, (Cn)_(x)-M13-reverse, (Cn)_(x)-T7, (Cn)_(x)-SP6, and (Cn)_(x)-T3.
 77. The kit of claim 75, wherein the chemically-enhanced primer is (Cn)_(x)-GSO, wherein GSO is a gene specific oligonucleotide sequence, and comprises 50 or fewer nucleotides.
 78. The kit of claim 74, further comprising at least one of instructions for use, a nuclease, a sufficient quantity of enzyme for a sequencing reaction or a fragment analysis reaction, buffer to facilitate the sequencing reaction or fragment analysis reaction, dNTPs, modified dNTPs, dNTP analogs and 7-Deaza-dGTP for strand extension during sequencing reaction or fragment analysis reaction, ddNTPs, a dye-label, loading solution for preparation of the sequenced material or fragment analysis material for electrophoresis, genomic DNA as a template control, a size marker to insure that materials migrate as anticipated in the separation medium, and a protocol and manual to educate the user and limit error in use. 